JP2014224120A - Combination of blys inhibition and anti-cd 20 agent for treatment of autoimmune disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel combination therapies involving BLyS or BLyS/APRIL inhibition and anti-CD20 agents for the treatment of autoimmune diseases.SOLUTION: BLyS antagonist is a Fc-fusion protein which can be a TACI-Fc-fusion protein comprising the extracellular domain of TACI or a functional fragment thereof, a BAFF-R-Fc-fusion protein comprising the extracellular domain of BAFF-R or a functional fragment thereof, or a BCMA-Fc-fusion protein comprising the extracellular domain of BCMA or a functional fragment thereof. Some of anti-CD20 agents contemplated include RITUXAN(R), ocrelizumab, ofatumumab (HuMax-CD20(R)), TRU-015, and DXL625, although any agent that binds to CD 20 may be suitable. The methods of the present invention reduce the levels of B cells in patients in need of such reduction, such as those suffering from autoimmune diseases.

Description

  The present invention relates to novel combination therapies including BLyS or BLyS / APRIL inhibitors and anti-CD20 agents for the treatment of autoimmune diseases.

  Lymphocytes are one of several populations of white blood cells; they specifically recognize and respond to foreign antigens. The three major classes of lymphocytes are B lymphocytes (B cells), T lymphocytes (T cells) and natural killer (NK) cells. B lymphocytes are cells involved in antibody production and provide humoral immunity. B cells mature in the bone marrow and leave the bone marrow and express antigen-binding antibodies on their cell surface. Native B cells first encounter an antigen whose membrane-bound antibody is specific, cells begin to divide rapidly, and their progeny differentiate into effector cells called memory B cells and plasma cells. Memory B cells continue to express membrane-bound antibodies that have a long life span and have the same specificity as the original parent cells. Plasma cells do not produce membrane-bound antibodies, but instead produce secreted forms of antibodies. Secretory antibodies are the main effector molecules of humoral immunity.

  A group of tumor necrosis factor (TNF) receptors found on the surface of B cells under various conditions is one of the cellular regulators of B cell function in the immune system. In particular, three TNF receptors: a transmembrane activator and CAML interacting factor (TACI), a B cell activator belonging to TNF family receptors (BAFF-R), and a B cell mature protein (BCMA) Is known to bind to one or both of TNF ligand-B lymphocyte stimulating factor (BLyS. Also known as BAFF, TALL-1, ztnf4 and THANK) and proliferation-inducing ligand (APRIL). . Specifically, TACI and BCMA are known to bind to both BLyS and APRIL, and BAFF-R binds to BLyS only.

  Various functions of BLyS, such as B cell costimulation, plasmablast and plasma cell survival, Ig class switch, enhancement of B cell antigen presenting cell function, survival of malignant B cells, development of B-1 cell function, T-1 stage A number of BLyS antagonists have been developed to block B cell development beyond, as well as, but not limited to, complete germinal center formation. Some of these molecules can also bind to APRIL and block the effects of APRIL on B cells and other components of the immune system (Dillon et al. (2006) Nat. Rev. Drug Dis. 5 , 235-246). Molecules that have been developed to affect B cell function by interfering with BLyS and / or APRIL binding include BLyS antibodies such as Lymphost-B (Berimumab) (Baker et al, (2003) Arthritis Rheum, 48, 3253. -3265 and WO 02/02641); receptor-extracellular domain / Fc domain fusion proteins, such as TACI-Ig, such as in one particular embodiment, atacicept (US Patent Application No. 20060034852), BAFF-R-Fc ( WO 05/0000351), and BCMA-Ig, or other fusion proteins that utilize the receptor extracellular domain. Further classes of BLyS antagonists include other molecules that rely on the ability to bind to BLyS, such as AMG 623, receptor antibodies and others, which block the binding to its receptor disclosed in WO 03/035846 and WO 02/16312 Of the molecule.

  The CD20 antigen (also called human B lymphocyte restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine et al. J. Biol Chent. 264 (19): 11282-11287 (1989); and Einfeld et al. EMBO J. 7 (3): 711-717 (1988)). Antigen is also expressed on more than 90% of B-cell non-Hodgkin lymphoma (NHL) (Anderson et al. Blood 63 (6): 1424-1433 (1984)), but hematopoietic stem cells, pro-B cells, normal It is not found on plasma cells or other normal tissues (Tedder et al. J. Immunol. 135 (2): 973-979 (1985)). CD20 is an early step or steps in the activation process for cell cycle initiation and differentiation (Tedder et al., Supra) and a possible function as a calcium ion channel (Tedder et al. J. Cell. Biochem. 14D: 195 (1990)).

  Given the expression of CD20 on B cells, this antigen may serve as a candidate for “targeting” these cells to treat autoimmune diseases. In essence, such targeting is summarized as follows: An antibody specific for the CD20 surface antigen of B cells is administered to the patient. These anti-CD20 antibodies specifically bind to the CD20 antigen of both B cells that produce normal and harmful autoantibodies (face-up); antibodies that bind to the CD20 surface antigen are responsible for the destruction of these B cells and Can lead to depletion. Regardless of the approach, the main objective is to destroy cells that produce autoantibodies; the specific approach is determined by the specific anti-CD20 antibody utilized and can therefore be used to target the CD20 antigen The approach can vary considerably.

  Rituximab (Rituxan®) antibody is a genetically engineered chimeric murine / human monoclonal antibody directed against the CD20 antigen. Rituximab is an antibody termed “C2B8” in US Pat. No. 5,736,137 (issued April 7, 1998) (Anderson et al.).

  Rituxan® is approved for the treatment of patients with relapsed or refractory, low grade or follicular, CD20 positive B-cell non-Hodgkin lymphoma. The in vitro mechanism of action studies demonstrates that Rituxan® binds to human complement and lyses the B lymphocyte cell line by complement dependent cytotoxicity (CDC) (Reff et al. Blood 83 (2): 435-445 (1994)). Furthermore, it has significant activity in assays for antibody-dependent cellular cytotoxicity (ADCC). More recently, Rituxan® has been shown to have an antiproliferative effect in a tritium thymidine incorporation assay and directly induce apoptosis (other anti-CD19 and CD20 antibodies are not) (Maloney et al. al. Blood 88 (10): 637a (1996)). A synergy between Rituxan and chemotherapy and toxins has also been observed experimentally.

  In particular, Rituxan® sensitizes drug-resistant human B-cell lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin, and ricin (Demidem et al., Cancer Chemotherapy & Radiopharmaceuticals 12 (3): 177-186 (1997)). In vivo preclinical experiments show that Rituxan® depletes B cells from cynomolgus monkey peripheral blood, lymph nodes and bone marrow, possibly through complement and cell-mediated processes (Reff et al Blood 83 (2): 435-445 (1994)).

  Patents and patent publications relating to the CD20 antibody include US Pat. Nos. 5,776,456; 5,736,137; 6,399,061 and 5,843,439, and US patent applications US 2002 / 0197255A1, US 2003 / 0021781A1, US 2003/0082172 A1, US 2003. / 0095963 A1, US 2003/0147885 A1 (Anderson et al.); US Pat. No. 6,455,043B1 and WO00 / 09160 (Grillo-Lopez, A.); WO00 / 27428 (Grillo-Lopez and White); WO0027433 (Grillo-Lopez and WO00 / 44788 (Braslawsky et al.); WO01 / 10462 (Rastetter, W.); WO01 / 10461 (Rastetter and White); WO01 / 10460 (White and Grillo-Lopez); US patent applications US2002 / 0006404 and WO02 / US Patent Application US2002 / 0012665 A1 and WO01 / 74388 (Hanna N.); US Patent Application US2002 / 0058029 A1 (Hanna, N.); US Patent Application US 2003/0103971 A1 (Hariharan and Hanna) US Patent Application US2002 / 0009444A1 and WO01 / 80884 (Grillo-Lopez, A.); WO01 / 97858 (White, C.); US2002 / 0128488A1 and WO02 / 34790 (Reff, M.); WO02 / 060955 (Braslawsky et al.); WO02 / 096948 (Braslawsky et al.); WO02 / 079255 (Reff and Davies); US Pat. No. 6,171,586 B1 and WO98 / 56418 (Lam et al.); WO98 / 58964 (Raju, S.); WO99 / 22764 (Raju, S.); WO99 / 51642, US Pat. No. 6,194,551B1, US Pat. No. 6,242,195B1, US Pat. No. 6,528,624 B1 and US Pat. No. 6,538,124 (Idusogie et al.); WO / 42072 (Presta, L.); WO00 / 67796 (Curd et al.); WO01 / 03734 (Grillo-Lopez et al.); US patent applications US2002 / 0004587A1 and WO01 / US Patent Application 2002/0197256 (Grewal, I); US Patent Application US2003 / 0157108 A1 (Presta, L.); US Patent Nos. 6,090,365B1, 6,287,537B1, 6,015,542, No. 77342 (Miller and Presta); US Pat. Nos. 5,843,398 and 5,595,721 (Kaminski et al.); US Pat. Nos. 5,500,362; 5,677,180; 5,721,108 and 6,120,767 (Robinson et al.); US Pat. No. 6,410,391 B1 (Raubitschek et al.); National Patent No. 6,224,866B1 and WO00 / 20864 (Barbera-Guillem, E.); WO01 / 13945 (Barbera-Guillem, E.); WO00 / 67795 (Goldenberg); US Patent Applications US 2003 / 01339301A1 and WO00 / 74718 ( WO / 76542 (Golay et al.); WO01 / 72333 (Wolin and Rosenblatt); US Pat. No. 6,368,596B1 (Ghetie et al.); US Patent Application US2002 / 0041847 A1 (Goldenberg, D.); US Pat. No. US2003 / 0026801A1 (Weiner and Hartmann); WO02 / 102312 (Engleman, E.); US Patent Application 2003/0068664 (Albitar et al.); WO03 / 002607 (Leung, S.); WO049694 (Wolin et al.); WO03 / 061694 (Sing and Siegall), each of which is incorporated herein by reference. See also US Pat. No. 5,849,898 and European Patent Application 330,191 (Seed et al.); US Pat. No. 4,861,579 and EP332,865A2 (Meyer and Weiss); USP 4,861,579 (Meyer et al.) And WO95 / 03770 (Bhat et al.).

  Publications on therapy with rituximab include: Perotta and Abuel “Response of chronic relapsing ITP of 10 years duration to Rituximab” Abstract # 3360 Blood 10 (1) (part 1-2): p. 88B (1998); Stashi et al. “Rituximab chimeric anti-CD20 monoclonal antibody treatment for adults with chronic idiopathic thrombocytopenic purpura” Blood 98 (4): 952-957 (2001); Mattews, R. “Medical Heretics” New Scientist ( 7 April, 2001); Leandro et al. “Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion” Ann Rheum Dis 61: 833-888 (2002); Leandro et al. “Lymphocyte depletion in rheumatoid arthritis: early evidence Arthritis and Rheumatism 44 (9): S370 (2001); Leandro etal. “An open study of B lymphocyte depletion in systemic lupus erythematosus”, Arthritis & Rheumatism 46 (1): 2673-2677 (for safety, efficacy and dose response. 2002); Edwards and Cambridge “Sustained improvement in rheumato id arthritis following a protocol designed to deplete B lymphocytes ”Rheumatology 40: 205-211 (2001); Edwards et al.“ B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders ”Biochem. Soc. Trans. 30 (4): 824-828 (2002); Edwards et al. “Efficacy and safety of Rituximab, a B-cell targeted chimeric monoclonal antibody: A randomized, placebo controlled trial in patients with rheumatoid arthritis. Arthritis and Rheumatism 46 (9): S197 (2002 ); Levine and Pestronk “IgM antibody-related polyneuropathies: B-cell depletion chemotherapy using Rituximab” Neurology 52: 1701-1704 (1999); DeVita et al. “Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis” Arthritis & Rheum 46: 2029-2033 (2002); Hidashida et al. “Treatment of DMARD-Refractory rheumatoid arthritis with rituximab.” Presented at the Anfzual Scientific Meeting of the American College of Rheumatology; Oct 24-29; New Orleans, LA 2002; Tuscano, J. “Successful treatment of Infliximab-refractory rheumatoid arthritis with rituximab ”Presented at the Annual Scientific Meeting of the American College of Rheumatology; Oct 24-29; New Orleans, LA 2002.

  One aspect of the present invention is a method for reducing B cell levels in a mammal, comprising administering a BLyS antagonist and an anti-CD20 agent. One preferred method is that the BLyS antagonist is a TACI-Fc fusion protein comprising the extracellular domain of TACI or a functional fragment thereof, a BAFF-R-Fc fusion protein comprising the extracellular domain of BAFF-R or a functional fragment thereof, or This is the case when the Fc fusion protein can be a BCMA-Fc fusion protein comprising the extracellular domain of BCMA or a functional fragment thereof. In particular, the Fc fusion protein comprises the polypeptide sequence of SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 26.

  In another embodiment, the BLyS antagonist is preferably a BLyS antibody that binds BLyS within the region comprising amino acids 162-275 of SEQ ID NO: 8, or a BLyS antibody known as LymphoStat-B. In a further embodiment, the BLyS antagonist is a TACI antibody that binds TACI within a region comprising 72-109 of SEQ ID NO: 2 or 82-222 of SEQ ID NO: 2.

  In the methods of the invention, some of the contemplated anti-CD20 agents include Rituxan®, but any agent that targets the CD20 antigen is suitable. The B cell level reduced by the method of the present invention is at least one of the measured values of total B cell count (eg circulating B cell count), memory B cell count and spleen B cell count (eg germinal center B cell count). Can be one.

  The invention also encompasses a method of alleviating a B cell regulatory autoimmune disorder, comprising administering a therapeutically effective amount of an anti-CD20 agent and a BLyS antagonist to a patient suffering from said disorder. In one embodiment, the autoimmune disorder is rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura ( ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuritis, myasthenia gravis, vasculitis, diabetes mellitus, Renault Selected from the group consisting of syndrome, Sjogren's syndrome and glomerulonephritis. One disease specifically treated in this way is systemic lupus erythematosus (SLE).

  In particular, where the autoimmune disorder is rheumatoid arthritis, systemic lupus erythematosus or lupus nephritis, in one embodiment, the BLyS antagonist and anti-CD20 agent are immunosuppressive drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs), It can be administered in further conjunction with therapy with glucocorticoids, prednisone and disease modifying anti-rheumatic drugs (DMARD).

  The methods of the invention may also utilize immunosuppressive drugs that have been shown to be effective in treating autoimmune diseases. Examples of immunosuppressive drugs intended for use in the methods of the invention are cyclophosphamide (CYC), azathioprine (AZA), cyclosporin A (CSA) and mycophenolate mofetil (MMF).

  In any of the methods of treating or alleviating a disorder where an anti-CD20 agent and a BLyS antagonist are administered to a patient, the anti-CD20 agent and the BLyS antagonist can be administered simultaneously or sequentially. In one particular embodiment, the anti-CD20 agent is administered before the BLyS antagonist.

  Also provided is a composition comprising an anti-CD20 agent and a BLyS antagonist.

  Further provided by the present invention is a product comprising an anti-CD20 agent, a BLyS antagonist and a label, wherein the label is for the composition to treat a B cell regulatory autoimmune disorder. It is a product to show.

  In any of the method, composition and product embodiments of the invention, when the BLyS antagonist or anti-CD20 agent is an antibody, it includes chimeric and humanized antibodies.

  In any of the method, composition and product embodiments of the invention, the BLyS antagonist, in one embodiment, is a fusion protein between the extracellular domain of a receptor that binds BLyS and the Fc domain of an immunoglobulin, or Fc fusion protein. In a particular embodiment, the Fc fusion protein is a TACI-Fc fusion protein comprising an extracellular domain of TACI, in particular atacicept, a BAFF-R-Fc fusion protein comprising an extracellular domain of BAFF-R, in particular BR3-Ig, and BCMA Selected from the group consisting of BCMA-Fc fusion proteins comprising the extracellular domain of In other embodiments, the BLyS antagonist is a BLyS antibody, in particular a BLyS antibody that binds BLyS within a region of BLyS comprising residues 162-275, particularly lymphostat B. In another embodiment, the BLyS antagonist binds at a region comprising residues 23-38 of a BAFF-R antibody, eg, human BAFF-R. In another embodiment, the BLyS antagonist is a TACI antibody, a BCMA antibody, or an antibody that binds both molecules as described in US Patent Application 2003-0012783.

FIG. 6 shows exemplary FACS results utilizing the gating strategy of Example 3 and subsequent analysis of B cell levels of cell populations, particularly in cynomolgus monkey spleen. A specific example of this is a spleen treated with vehicle instead of a control, for example Rituxan®. This example shows total spleen lymphocytes (FIG. 1A), CD40 + / CD3-B lymphocytes (FIG. 1B), and CD40 + / CD3- / CD21 + / CD27 + memory B cells (FIG. 1C). The results reported in Example 4 are average values of FACS analysis performed in the same manner as the example shown here.

6 is a graph showing the absolute number of total B cells (CD45 + / B220 +) in mice throughout the 80 days of testing for the combination experiment described in Example 5. FIG.

6 is a graph showing the absolute number of human CD20 + B cells (B220 + / huCD20 +) in human CD20 transgenic mice throughout the 80 days of testing for the combination experiment described in Example 5. FIG.

7 shows exemplary FACS results utilizing the gating strategy of Example 5 for analysis of cell populations, particularly B cell levels in mouse peripheral blood injections. A specific example of this is a human CD20 transgenic mouse treated with a control, for example a vehicle. In this example, FIG. 4A shows total leukocytes that are selected for CD45 lymphocytes, and then in FIG. 4B, CD45 + / B220 + B cells are selected from the CD45 + lymphocyte population. FIG. 4C shows the selection for lymphocytes from peripheral blood, FIG. 4D is a graph showing the selection of B220 plus and CD3 + cells from the lymphocyte population, and FIG. 4E shows the majority of the B220 + lymphocyte population. It shows that it is huCD20 +. Detailed Description of Preferred Embodiments

  Although anti-CD20 agent treatment appears to be useful for the treatment of autoimmune diseases, administration of a combination of anti-CD20 agent and a BLyS antagonist results in multiple signals in B cells that are thought to be involved in the production of antibodies directed against the self-antigen. It has been discovered from the experiments herein that this is a therapeutic method that blocks the pathway, thereby triggering an autoimmune condition and perpetuating it. This results in a decrease in the number of B cells and, in turn, a decrease in circulating immunoglobulin in mammals undergoing such treatment. Considered without being bound by theory, as such, circulating immunoglobulins are at least partially involved in triggering negative symptoms of autoimmune disease and are therefore directed against anti-CD20 agents and the BLyS pathway. Both combinations provided provide a novel method for the treatment of B cell mediated diseases such as B cell based autoimmune diseases. Combination therapy with anti-CD20 agents and BLyS antagonists may provide a more effective alternative to current treatments for certain diseases, such as SLE.

  An “autoimmune disease” as used herein is any malignant disease or disorder resulting from an antibody directed against an individual's own (self) antigen and / or tissue.

  As used herein, “B cell depletion” refers to a reduction in B cell levels in an animal or human after drug or antibody treatment as compared to pre-treatment levels. B cell levels can be measured using well-known assays, eg, by obtaining complete blood counts, by FACS analysis staining for known B cell markers, and by methods as described in the experimental examples. B cell depletion can be partial or complete. In patients taking B cell depleting drugs, B cells are generally depleted with respect to the duration that the drug is circulating in the patient's body and the time for B cell recovery.

  The term “anti-CD20 agent” encompasses any molecule that binds to CD20 and, in the most preferred embodiment, targets cells associated with the CD20 protein for killing. Such molecules include anti-CD20 antibodies, such as Rituxan®, as well as subsequent versions of the drug, such as ocrelizumab, a humanized version of the antibody, offatumbub (HuMax-CD20® fully human anti-CD20 agent) , DXL625 (second generation anti-CD20 monoclonal), GA101 (third generation anti-CD20 agent with Fc region alteration), anti-CD20 molecule described in US patent application 20060121032, anti-CD20 molecule described in US patent application 20070014720, Anti-CD20 molecule described in US patent application 20060251652, anti-CD20 molecule described in US patent application 20050069545, anti-CD20 molecule described in US patent application 200040167319, TRU-015 (small molecule immunopharmaceutical molecule targeting CD20 ), As well as ibritumomab (zevalin (registered trader) )) Include conjugated molecules such as.

  An “immunosuppressive drug” is any molecule that interferes with the immune system and blunts its response to foreign or self antigens. Cyclophosphamide (CYC) and mycophenolate mofetil (MMF) are two such types of molecules. The term is intended to encompass any agent or molecule that is useful as a therapeutic agent in down-regulating the immune system. This method is particularly useful for autoimmune diseases such as rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura. (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuritis, myasthenia gravis, vasculitis, diabetes mellitus, Contemplates drugs that have been used to treat Lehno syndrome, Sjogren's syndrome and glomerulonephritis.

  The terms “BLyS” or “BLyS polypeptide”, “TALL-1” or “TALL-1 polypeptide”, or “BAFF” or “BAFF polypeptide” as used herein refer to the “native sequence BLyS”. It includes "polypeptide" and "BLyS variant". “BLyS” is the designation given to the polypeptide encoded by the human BLyS sequence (SEQ ID NO: 7) or mouse BLyS sequence (SEQ ID NO: 9). Polypeptides that exhibit BLyS biological activity are also encompassed within this designation. For example, biologically active BLyS enhances one or a combination of the following events in vitro or in vivo: increased B cell survival, increased levels of IgG and / or IgM, plasma cells As well as the processing of NF-Kb2 / 100 to p52NF-Kb in splenic B cells (eg, Batten, M et al., (2000) J. Exp. Med. 192: 1453-1465; Moore, et al., (1999) Science 285: 260-263; Kayagaki, et al., (2002) 10: 515-524). Several assays useful for testing BLyS antagonists are well known to those skilled in the art, such as the B cell proliferation assay described, inter alia, in WO 00/40716.

In short, human B cells are isolated from peripheral blood mononuclear cells using CD19 magnetic beads and VarioMacs magnetic separation system (Miltenyi Biotec Auburn, CA) according to the manufacturer's instructions. Purified B cells are mixed with soluble BLyS (25 ng / ml) and recombinant human IL-4 (10 ng / ml, Pharmingen), and cells are plated on round bottom 96 well plates at 1 × 10 ⁵ cells / well. Plated. The BLyS antagonist to be tested is diluted from about 5 μg / ml to about 6 ng / ml, incubated with B cells for 5 days, and pulsed overnight at 1 μCi ³ H-thymidine / well on day 4. As a control, BLyS antagonists can also be incubated with B cells and IL-4 without BLyS. Plates are harvested using a Packard plate harvester and counted using a Packard reader.

  A “native sequence” polypeptide includes a polypeptide having the same amino acid sequence as the corresponding polypeptide from nature. Such native sequence polypeptides can be isolated from nature and produced by recombinant and / or synthetic means. The term “native sequence” specifically includes naturally truncated, soluble or secreted forms (eg, extracellular domain sequences), naturally occurring mutant forms (eg, alternatively spliced forms), as well as naturally occurring, of a polypeptide. Includes allelic variants.

  In general, a “variant” polypeptide with respect to any of the polypeptides disclosed herein has one or more amino acid residues at the N and / or C terminus of the full length or “native sequence” amino acid sequence. As well as polypeptides that are added or deleted within one or more internal domains. When considering the extracellular domain of a receptor, fragments that bind the native sequence BLyS polypeptide are also contemplated. Conversely, when considering BLyS fragments, fragments that bind any one or more of the three BLyS receptors are contemplated. Usually, a variant polypeptide has at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acids with the polypeptide or a specific fragment thereof. Sequence identity, more preferably at least about 83% amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86% Amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89% amino acid sequence identity, more preferably at least about about 90% amino acid sequence identity, more preferably at least about 91% amino acid sequence identity, more preferably at least about 92% amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably At least about 95% amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98% amino acid sequence identity, Preferably it has at least about 99% amino acid sequence identity. In general, variant polypeptides do not include a native polypeptide sequence. Usually, variant polypeptides are at least about 10 amino acids long, often at least about 20 amino acids long, more often at least about 30 amino acids long, more often at least about 40 amino acids long, more often at least about 50 amino acids long, more often at least about at least about 60 amino acids long, more often at least about 70 amino acids long, more often at least about 80 amino acids long, more often at least about 90 amino acids long, more often at least about 100 amino acids long, more often at least about 150 amino acids long, more often at least about 200 amino acids long Long, more often at least about 250 amino acids long, more often at least about 300 amino acids long, or more.

  As described above, BLyS antagonists can function directly or indirectly to partially or completely block, suppress or neutralize BLyS in vitro or in vivo. For example, a BLyS antagonist can bind BLyS directly. For example, the direct binding agent is a polypeptide comprising the extracellular domain (ECD) of a BLyS receptor such as TACI, BAFF-R and BCMA.

  The BLyS receptor involved in the present invention can be described as follows. The TACI polypeptides of the present invention include TACI polypeptides comprising or consisting of amino acids 1-246 of SEQ ID NO: 2. The general term “TACI” encompasses the TACI polypeptides described in WO 98/39361, WO 00/40716, WO 01/85782, WO 01/87979, WO 01/81417 and WO 02/094852. The TACI polypeptides of the invention can be isolated from a variety of sources, such as from human tissue types, or from another source, or can be prepared by recombinant and / or synthetic methods. The BAFF-R polypeptide of the present invention includes a BAFF-R polypeptide comprising or consisting of a contiguous sequence of amino acid residues 1 to 184 of SEQ ID NO: 4. The general term “BAFF-R” encompasses the BAFF-R polypeptides described in WO 02/24909 and WO 03/14294. The BAFF-R polypeptides of the invention can be isolated from a variety of sources, such as from human tissue types, or from another source, or can be prepared by recombinant and / or synthetic methods. BCMA polypeptides of the present invention include BCMA polypeptides comprising or consisting of amino acid residues 1 to 184 of SEQ ID NO: 6. The general term “BCMA” is described in Laabi et al., EMBO J. 11: 3897-3904 (1992); Laabi et al., Nucleic Acids Res., 22: 1147-1154 (1994); Gras et al., Int. Immunology, 7: 1093-1106 (1995); and BCMA described in Madry et al., Int. Immunology, 10: 1693-1702 (1998). The BCMA polypeptides of the invention can be isolated from a variety of sources, such as from human tissue types, or from another source, or can be prepared by recombinant and / or synthetic methods.

  For the purpose of functioning as a BLyS antagonist, the ECD of these receptors is a polypeptide that has essentially no transmembrane or cytoplasmic domain that generally retains the ability to bind BLyS. Specifically, the extracellular domain of TACI can comprise amino acids 1-154 of the TACI polypeptide sequence (SEQ ID NO: 2). Furthermore, ECD has been described in fragments or variants of this sequence such as von Bulow et al., Supra, WO 98/39361, WO 00/40716, WO 01/85782, WO 01/87979 and WO 01/81417. It can be an ECD form of TACI. In particular, these ECD forms are amino acids 1 to 106 of SEQ ID NO: 2, amino acids 1 to 142 of SEQ ID NO: 2, amino acids 30 to 154 of SEQ ID NO: 2, amino acids 30 to 106 of SEQ ID NO: 2, amino acids 30 of SEQ ID NO: 2 -110, amino acids 30-119 of SEQ ID NO: 2, amino acids 1-166 of SEQ ID NO: 2, amino acids 1-165 of SEQ ID NO: 2, amino acids 1-114 of SEQ ID NO: 2, amino acids 1-119 of SEQ ID NO: 2, SEQ ID NO: 2 amino acids 1-120 and amino acids 1-126 of SEQ ID NO: 2. Furthermore, the TACI ECD can include molecules with only one rich cysteine domain.

  EFF forms of BAFF-R include those comprising amino acids 1 to 71 of the BAFF-R polypeptide sequence (SEQ ID NO: 4). Furthermore, the ECD can be a fragment or variant of this sequence, for example the ECD form of BAFF-R as described in WO 02/24909, WO 03/14294 and WO 02/38766. In particular, these ECD forms are amino acids 1 to 77 of SEQ ID NO: 4, amino acids 7 to 77 of SEQ ID NO: 4, amino acids 1 to 69 of SEQ ID NO: 4, amino acids 7 to 69 of SEQ ID NO: 4, amino acid 2 of SEQ ID NO: 4 ~ 62, amino acids 2 to 71 of SEQ ID NO: 4, amino acids 1 to 61 of SEQ ID NO: 4 and amino acids 2 to 63 of SEQ ID NO: 4, amino acids 1 to 45 of SEQ ID NO: 4, amino acids 1 to 39 of SEQ ID NO: 4, SEQ ID NO: 4 amino acids 7-39, amino acids 1-17 of SEQ ID NO: 4, amino acids 39-64 of SEQ ID NO: 4, amino acids 19-35 of SEQ ID NO: 4, and amino acids 17-42 of SEQ ID NO: 4. In addition, the BAFF-R ECD can include molecules with one rich cysteine domain.

  BCMA ECD forms include those comprising amino acids 1-48 of the BCMA polypeptide sequence (SEQ ID NO: 6). Further, the ECD can be a fragment or variant of this sequence, for example the ECD form of BCMA as described in WO 00/40716 and WO 05/075511. In particular, these ECD forms are amino acids 1-150 of SEQ ID NO: 6, amino acids 1-48 of SEQ ID NO: 6, amino acids 1-41 of SEQ ID NO: 6, amino acids 8-41 of SEQ ID NO: 6, amino acids 8 of SEQ ID NO: 6 37, amino acids 8 to 88 of SEQ ID NO: 6, amino acids 41 to 88 of SEQ ID NO: 6, amino acids 1 to 54 of SEQ ID NO: 6, amino acids 4 to 55 of SEQ ID NO: 6, amino acids 4 to 51 of SEQ ID NO: 6, and SEQ ID NO: 6 amino acids 21-53 may be included. In addition, BCMA ECD may include molecules with only a partially rich cysteine domain.

  In a further embodiment, the BLyS binding region of the BLyS receptor (eg, the extracellular domain of BAFF-R, BCMA, or TACI or a fragment thereof) is fused to the Fc portion of an immunoglobulin molecule and its solubility in vivo. Can help. According to one embodiment, the BLyS antagonist binds to a BLyS polypeptide with a binding affinity of 100 nM or less. According to another embodiment, the BLyS antagonist binds to a BLyS polypeptide with a binding affinity of 10 nM or less. According to yet another embodiment, the BLyS antagonist binds to a BLyS polypeptide with a binding affinity of 1 nM or less.

  In another example, a BLyS antagonist includes a BLyS binding polypeptide that is not a native sequence or a variant thereof. Some examples of such polypeptides are those having the sequence of Formula I, Formula II, Formula III as described in WO 05/000351. In particular, some binding polypeptides include ECFDLLVRAWVPCSVVLK (SEQ ID NO: 13), ECFDLLVRHWVPCGLLR (SEQ ID NO: 14), ECFDLLVRRWVPCEMLG (SEQ ID NO: 15), ECFDLLVRSWVPCHMLLR (SEQ ID NO: 16), ECFDLLVRWRWG51 Includes the sequences listed in FIG.

  Alternatively, a BLyS antagonist binds the extracellular sequence of the native sequence TACI, BAFF-R or BCMA at its BLyS binding region to partially or fully inhibit BLyS binding in vitro, in situ or in vivo. Can block, inhibit, or neutralize. For example, such an indirect antagonist is a TACI antibody that binds in a region of TACI such that BLyS binding is sterically hindered. For example, binding at amino acids 72-109 or adjacent regions is thought to block BLyS binding. It is also beneficial to block APRIL binding with this molecule, which is thought to occur in the region of amino acids 82-222. Another BLyS antagonist is a BAFF-R antibody that binds in the region of BAFF-R such that the binding between human BAFF-R and BLyS is sterically hindered. For example, binding at amino acids 23-38 or amino acids 17-42 or adjacent regions is thought to block BLyS binding. Finally, a further indirect antagonist is a BCMA antibody that binds in a region of BCMA so that BLyS binding is sterically hindered. For example, amino acids 5-43 or adjacent regions are thought to block BLyS (or APRIL) binding.

  In some embodiments, BLyS antagonists according to the present invention include BLyS antibodies. The term “antibody” is used in its broadest sense when referring to and specifically encompasses, for example, monoclonal antibodies, polyclonal antibodies, antibodies with multiple epitope specificity, single chain antibodies, and antibodies. According to some embodiments, the polypeptides of the invention may be, for example, in the variable region or in the CDRs so that the antibody binds to TACI, BAFF-R or BCMA and can inhibit BLyS binding to them. It is fused to the antibody framework or suppresses BLyS signaling. An antibody comprising a polypeptide of the invention can be a chimeric, humanized or human antibody. An antibody comprising a polypeptide of the invention can be an antibody fragment. Alternatively, the antibodies of the present invention can be produced by immunizing an animal with a polypeptide of the present invention. Accordingly, antibodies directed against the polypeptides of the present invention are contemplated.

  In particular, one of human BLyS (SEQ ID NO: 8) comprising residues 162-275 of amino acids selected from the group consisting of 162, 163, 206, 211, 231, 233, 264 and 265 of human BLyS and / or adjacent amino acids. Antibodies specific for BLyS that bind within the region are contemplated. As a result of antibody binding, the antibody sterically hinders BLyS binding to one or more of its receptors. Such antibodies are described in WO 02/02641 and WO 03/055979. A particularly preferred antibody is that described as Lymphhost B (Baker et al. (2003) Arthritis Rheum, 48, 3253-3265).

  The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, individual antibodies comprising that population may be present in small amounts. Identical except for some natural mutations.

  Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Directed. In addition to their specificity, monoclonal antibodies are beneficial in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (eg, US Pat. No. 4,816,567). No.). “Monoclonal antibody” is a technique described in, for example, Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). Can also be used to isolate from a phage antibody library.

  As monoclonal antibodies herein, specifically, a portion of the heavy and / or light chain is derived from a particular species or is identical to the corresponding sequence in an antibody belonging to a particular antibody class or subclass. While the remaining chain (s) are identical or homologous to the corresponding sequence in the recession from another species or belonging to another antibody class or subclass. “Chimeric” antibodies (immunoglobulins), as well as fragments of such antibodies as long as they exhibit the desired biological activity (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA). 81: 6851-6855 (1984)). Methods for producing chimeric antibodies are known in the art.

  “Humanized” forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′, F (ab ′)) that contain minimal sequence derived from non-human immunoglobulin. 2 or other antigen-binding subsequence of the antibody).

  For the most part, humanized antibodies are non-human species (donor antibodies) in which residues from the complementarity determining regions (CDRs) of the recipient have the desired specificity, affinity and ability, eg, mouse, rat or Human immunoglobulin (recipient antibody) that is replaced by residues from the rabbit CDRs. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences. These modifications are made to further improve and maximize antibody performance. In general, a humanized antibody is one in which the humanized antibody comprises substantially all of at least one, typically two variable domains, wherein all or substantially all of the hypervariable loops are non-human. Although corresponding to that of an immunoglobulin and all or substantially all of the FR region is of a human immunoglobulin sequence, the FR region includes one or more amino acid substitutions that improve binding affinity. obtain. The number of these amino acid substitutions in the FR is typically 6 or less in the H chain and 3 or less in the L chain. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2 : See 593-596 (1992). Humanized antibodies include PRIMATIZED antibodies in which the antigen-binding region of the antibody is derived from an antibody produced, for example, by immunizing a macaque monkey with an extra-sugar antigen. Methods for producing humanized antibodies are known in the art.

  Human antibodies can also be produced using various techniques known in the art, such as phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al. , J. Mol. Biol., 222: 581 (1991)). Techniques such as Cole et al. And Boerner et al. Are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991)).

  “Functional fragments” of the binding antibodies of the invention retain binding to BLyS, TACI, BAFF-R or BCMA with substantially the same affinity as the intact full chain molecule from which they are derived, and A fragment that can deplete B cells as measured by in vitro or in vivo assays as described herein.

  Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors ( Down-regulation (eg B cell receptor); and B cell activation.

  “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” is bound on Fc receptors (FcR) present on certain cytotoxic cells, such as natural killer (NK) cells, neutrophils and macrophages. Secretory Ig refers to a form of cytotoxicity that specifically binds these cytotoxic effector cells to antigen-bearing target cells and then kills the target cells with cytotoxins. The antibody “embraces” the cytotoxic cells and is unconditionally required for such killing. NK cells, the primary cell for mediating ADCC, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Ann. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of the molecule, an in vitro ADCC assay as described in US Pat. Nos. 5,500,362 or 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule can be assessed in vivo in an animal model such as disclosed for example in Clynes etal. PNAS (USA) 95: 652-656 (1998). .

  “Complement dependent cytotoxicity” or “CDC” refers to lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the primary component of the complement system (Clq) to antibodies (of the appropriate subclass) that are bound to their cognate antigens. In order to assess complement activation, a CDC assay as described, for example, in Gazzano-Santoroetal., J. Immunol. Methods 202: 163 (1996) can be performed.

  An “isolated” antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that prevent diagnostic or therapeutic use for antibodies, examples include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is (1) more than 95% by weight as determined by the Raleigh method, most preferably more than 99% by weight, and (2) at least 15 residues of N by use of a spinning cup sequenator. Purified to a degree sufficient to obtain terminal or internal amino acid sequences, or (3) Coomassie blue, or preferably by SDS-PAGE, preferably under reducing or non-reducing conditions using silver staining .

  Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

  Amino acids can be grouped by similarity in their side chain properties (AL Lehninger, in Biochemistry, second ed., Pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) (2) Uncharged polarity: Gly (G) , Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidity: Asp (D), Glu (E) (4) Basicity: Lys (K), Arg (R), His (H-). Alternatively, natural residues may be grouped based on common side chain properties: (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidity: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

  The term “conservative” amino acid substitution, as used within the present invention, is intended to refer to an amino acid substitution that substitutes a functionally equivalent amino acid. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids of similar polarity can act as functional equivalents, producing silent changes within the amino acids of the peptide. In general, substitutions within a group can be considered conservative with respect to structure and function. However, those skilled in the art will recognize that the role of a particular residue is determined by its context within the three-dimensional structure of the molecule in which it occurs. For example, Cys residues occur in the oxidized (disulfide) form, which is less polar than the reduced (thiol) form. The long aliphatic part of the Arg side chain may constitute an important feature of its structural or functional role, which is best conserved by substitution of a nonpolar residue rather than another basic residue. obtain. Furthermore, it is recognized that side chains containing aromatic groups (Trp, Tyr and Phe) may be involved in ion-aromatic or “cation-pi” interactions. In these cases, substitution of one of these side chains by a member of an acidic or uncharged polar group may be conservative with respect to structure and function. Residues such as Pro, Gly, and Cys (disulfide form) can directly affect the main chain conformation and often cannot be substituted without structural distortion.

  “Percent amino acid sequence identity” with respect to the ligand or receptor polypeptide sequences identified herein is the alignment of sequences, introducing gaps and, where necessary, up to% sequence identity. Amino acid residues in a candidate sequence that are identical to such a ligand or receptor sequence identified herein after achieving the above and not considering any conservative substitutions as part of sequence identity Is defined as the percentage. Alignment for the purpose of determining% amino acid sequence identity can be done in various ways within the art, such as public, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalin (DNASTAR) software. Can be achieved using computer software available in Using any algorithm required to achieve maximal alignment over the entire length of the sequences being compared, one skilled in the art can determine appropriate parameters for measuring alignment. For purposes herein, however,% amino acid sequence identity values are obtained as follows by using the sequence comparison computer program ALIGN-2, in which case the complete sequence for the ALIGN-2 program: The source code is presented in the table below. The ALIGN-2 sequence comparison computer program was created by Genentech, and the source code shown in the table below is filed with user documentation at the US Copyright Office (Washington DC, 20559), where: It is registered under US copyright registration number TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, California) or can be compiled from the source code presented in the table below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

  A useful method for the identification of certain residues or regions in a protein that are preferred positions for mutagenesis is described in “Alanine Scanning, as described by Cunningham and Wells Science, 244: 1081-1085 (1989). It is called “mutagenesis”. A group of single residues or target residues is identified (eg charged residues such as arg, asp, his, lys and glu) and replaced by neutral or negatively charged amino acids (most preferably alanine or polyalanine) Influencing the interaction between the amino acid and the binding target. The amino acid positions that demonstrate functional sensitivity to the substitution are then refined by introducing further or other variants at or for the substitution site. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the outcome of a mutation at a given site, a la scanning or random mutagenesis is performed at the target codon or region and the expression variants are screened for the desired activity.

  The term “dihedral angle” refers to rotation around a single bond (see, eg, Creighton, T.E., (1993) Protein: Structures and Molecular Properties, 2 ed., W.H. Freeman and Company, New York, NY). The term “phi (φ)” is a dihedral angle meaning rotation around the N—C bond of an amino acid (eg, Creighton, TE, (1993) Protein: Structures and Molecular Properties, 2 ed., WH Freeman and Company, New York, NY). Type I β-rotation is described in Hutchinson, E.G. & Thornton, J.M. (1994) A revised set of potentials for beta burn formation in proteins. Protein Science 3, 2207-2216.

  “Fusion protein” and “fusion polypeptide” refer to a polypeptide having two parts covalently linked together, wherein each of the parts is a polypeptide having different properties. The property can be a biological property, such as activity in vitro or in vivo. A property can also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, and the like. The two moieties can be linked directly by a simple peptide bond or via a peptide linker containing one or more amino acid residues. In general, the two moieties and the linker are in reading frame with each other.

  “Conjugate” refers to any hybrid molecule, such as a fusion protein, and a molecule containing both amino acid or protein and non-protein portions. Conjugates can be synthesized by various techniques known in the art, such as recombinant DNA techniques, solid phase synthesis, liquid phase synthesis, organic chemical synthesis techniques, or a combination of these techniques. The choice of synthesis depends on the particular molecule to be generated. For example, hybrid molecules that are not completely “protein” in nature can be synthesized by a combination of recombinant and liquid phase techniques.

  As used herein, the term “Fc fusion protein” refers to an antibody-like molecule that combines the binding specificity of a heterologous protein with the effector function of an immunoglobulin constant domain. Structurally, an Fc fusion protein comprises a fusion of an amino acid sequence and an immunoglobulin display domain sequence having a desired specificity other than the antigen recognition and binding site of an antibody (ie, “heterologous”). An Fc fusion protein molecule typically includes a contiguous amino acid sequence comprising at least a receptor or ligand binding site. The immunoglobulin constant domain sequence in the Fc fusion protein can be any immunoglobulin, eg, IgG-1, IgG-2, IgG-3 or IgG-4 subtype, IgA (eg, IgA-1 and IgA-2), IgE, Obtained from IgD or IgM. For example, a useful Fc fusion protein according to the present invention is a polypeptide comprising a BLyS binding portion of a BLyS receptor without the transmembrane or cytoplasmic sequence of the BLyS receptor. In one embodiment, the extracellular domain of BAFF-R, TACI or BCMA is fused to a constant domain of an immunoglobulin sequence.

  The term “mammal” refers to mammals such as humans, farm animals and farm animals, and any animals classified as zoo animals, sport animals or pet animals such as dogs, horses, cats, cows and the like. Preferably the mammal is a human.

  The term “therapeutically effective amount” refers to an amount of an antibody or antagonist agent effective to “reduce” or “treat” a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of the drug reduces the number of cancer cells, reduces tumor size, suppresses cancer cell invasion to peripheral organs (ie slows to some extent, preferably stops), Tumor metastasis can be suppressed (slow to a certain extent, preferably stopped), tumor growth can be suppressed to a certain extent, and / or one or more of cancer-related symptoms can be alleviated to some extent (see “ See definition of “treated”). To the extent that the agent can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic.

  The BLyS or BLyS receptor antibodies of the invention can be produced by transiently or stably transfected eukaryotic host cells, such as CHO cells.

A “carrier”, as used herein, is a pharmaceutically acceptable carrier, excipient or stable that is cytotoxic to cells or mammals exposed to it alone at the dosage and dose used. Includes agents. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins For example, serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrin; Agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween polyethylene glycol (PEG) and Pluronics ™.
Polynucleotide, vector, host cell

  According to a number of embodiments disclosed herein, a BLyS antagonist can include a specific polypeptide that is produced using a specific polynucleotide in a specific vector and with a specific host cell. Various types of polypeptides of the invention have been extensively described and can be selected from the group consisting of receptor-based sequences, antibody-based sequences, and artificial (ie non-native) binding sequences. Examples of receptor-based sequences are those that bind BLyS in vivo, such as sequences that bind to BLyS isolated from or derived from the domain of a receptor such as TACI, BAFF-R or BCMA. Antibody-based sequences are produced using antibody development techniques and retain the general structure of an antibody molecule. An example of an antibody-based fly is an antibody against a receptor for lymphostatin B or BLyS. Examples of artificial binding sequences include 17mer peptides described herein, polypeptides that incorporate one or more 17mers as a core region, and covalently modified forms of 17mer peptides and polypeptides (eg, Fc fusions). Protein, labeled polypeptide, protected polypeptide, conjugated polypeptide, fusion protein, etc.). Various techniques used to produce these forms of polypeptides are described herein. Methods for labeling polypeptides and coupling molecules to polypeptides are known in the art.

  The compositions of the present invention can be prepared using recombinant techniques known in the art.

  The following relates to methods for producing such specific polypeptides by culturing host cells transformed or transfected with a vector containing the encoding nucleic acid and recovering the polypeptide from the cell culture (eg, See Sambrook et al., Molecular Cloning: A Laboratory Manual (New Y: Cold Spring Harbor Laboratory Press, 1989); Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

  Nucleic acid (eg, cDNA or genomic DNA) encoding the desired polypeptide can be inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. Various vectors are publicly available. Vector components generally include the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, each of which is described in a pond One or more of these may be mentioned, but are not limited to these. Any signal sequence, origin of replication, marker gene, enhancer element and transcription termination sequence that can be used are known in the art and are described in more detail in WO 97/25428.

  Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the encoding nucleic acid sequence. Promoters are located upstream (5 ') to the start codon (generally within about 100-1000 bp) of a structural gene that controls the transcription and translation of the particular nucleic acid sequence to which they are operably linked. Untranslated sequence. Such promoters are typically divided into two classes: inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients, or changes in temperature. At this point, a number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to the coding DNA by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector.

  Construction of a suitable vector containing one or more of the above components uses standard ligation techniques. The isolated plasmid or DNA fragment is cut, adapted and religated in the form desired to produce the required plasmid. The ligation mixture can be used to transform E. coli K12 strain 294 (ATCC 31,446) for analysis to confirm the correct sequence in the constructed plasmid, and successful transformants can be In some cases, it can be selected by ampicillin or tetracycline resistance. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and / or sequenced by standard techniques known in the art [eg Messing et al., Nucleic Acids Res., 9: 309 ( 1981); Maxam et al., Methods in Enzymology, 65: 499 (1980)].

  Expression vectors that provide for transient expression of the encoded DNA in mammalian cells can be used. In general, transient expression replicates efficiently in the host cell so that the host cell accumulates multiple copies of the expression vector and then synthesizes the high level of the desired polypeptide encoded by the expression vector. Including the use of the resulting expression vector [Sambrook et al., Supra]. Transient expression systems, including appropriate expression vectors and host cells, provide convenient positive identification of polypeptides encoded by cloned DNA, as well as rapid expression of such polypeptides with respect to desired biological or physiological properties. To enable effective screening.

  Other methods, vectors and host cells suitable for adapting the synthesis of the desired polypeptide in recombinant vertebrate cell culture are described in Gething et al., Nature, 293: 620-625 (1981); Mantei et al ., Nature, 281: 40-46 (1979); EP 117, 060; and EP 117,058.

  Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria such as Gram negative or Gram positive bacteria such as Enterobacteriaceae such as E. coli, Escherichia such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, and Bacillus, such as Bacillus subtilis and Bacillus licheniformis (eg, DD 266,710 (published on April 12, 1989)) Bacillus licheniformis 41P), Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces, but are not limited thereto. Preferably the host cell should secrete a minimal amount of proteolytic enzyme.

  In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors. Suitable host cells for the expression of glycosylated polypeptide are derived from multicellular organisms. Examples of all such host cells are further described in WO 97/25428.

  Host cells are transfected with the above expression or cloning vectors, preferably transformed, and suitable for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In a nutrient medium modified to be

Transfection refers to the uptake of an expression vector by a host cell, whether or not any coding sequence is actually expressed. Numerous methods of transfection are known to those skilled in the art, such as CaPO ₄ and electroporation. Successful transfection is generally recognized when any instruction for the operation of this vector occurs in the host cell.

  Transformation means introducing DNA into an organism so that the DNA can replicate as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. Calcium treatment with calcium chloride (described in Sambrook et al. (Supra)) or electroporation is commonly used for prokaryotes or other cells that contain a substantial cell wall barrier. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells (Shaw et al., Gene, 23: 315 (1983) and WO 89/05859 (published June 29, 1989)) Described in). Furthermore, plants can be transfected using sonication (described in WO 91/00358 (published 10 January 1991)).

  For mammalian cells that do not have such cell walls, the calcium phosphate precipitation method (Graham and van der Eb, Virology, 52: 456-457 (1978)) can be used. General aspects of mammalian cell host system transformation are described in US Pat. No. 4,399,216. Transformation in yeast is typically performed by Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: Performed according to the method of 3829 (1979). However, other methods for introducing DNA into cells, such as nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations such as polybrene, polyornithine can also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (1988). I want.

  Prokaryotic cells can be cultured in a suitable medium as generally described in Sambrook et al. (Supra). Examples of commercially available media include Ham F10 media (Sigma), minimal essential media (“MEM”, Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium (“DMEM”, Sigma). Any such medium can contain hormones and / or other growth factors (eg, insulin, transferrin or epidermal growth factor), salts (eg, sodium chloride, calcium, magnesium and phosphate), buffers, if necessary. (Eg, HEPES), nucleosides (eg, adenosine and thymidine), antibiotics (eg, gentamicin), trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar range), and glucose or equivalent Can be supplemented with energy sources. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used for the host cell selected for expression and will be apparent to those skilled in the art.

  In general, principles, protocols and practical techniques for maximizing the productivity of mammalian cell culture can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991).

  The expressed polypeptide can be recovered from the medium as a secreted polypeptide, but can also be recovered from the host cell lysate if produced directly without a secretory signal. If the polypeptide is membrane bound, it can be released from the membrane using a suitable detergent solution (eg, Triton X100) or its extracellular region can be released by enzymatic cleavage.

If the polypeptide is produced in a recombinant cell other than that of human origin, it does not have a protein or polypeptide of human origin. However, to obtain a preparation that is substantially homogeneous, it is usually necessary to recover or purify the polypeptide from recombinant cell proteins or polypeptides. As a first step, the medium or lysate is centrifuged to remove particulate cell debris. The following are examples of suitable purification techniques: fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica gel or a cation exchange resin such as DEAE; isoelectric point chromatography SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and by Protein A Sepharose column to remove contaminants such as IgG.
Phage display

  According to some embodiments, the following groups: Formula I, Formula II, Formula III, ECFDLLVRAWVPCSVLK (SEQ ID NO: 13), ECFDLLVRHWVPCGLLR (SEQ ID NO: 14), ECFDLLVRRWVPCEMLG (SEQ ID NO: 15), ECFDLLVRSWVPCHMLLR (SEQ ID NO: 16) A polypeptide of the present invention selected from ECFDLLVRHWVACGLLR (SEQ ID NO: 17) and the sequence listed in FIG. 32 of WO 05/000351 may be utilized for phage display.

  The use of phage display technology allows for the generation of large libraries of protein variants that can be rapidly classified with respect to their sequences that bind target molecules with high affinity. Nucleic acid encoding a variant polypeptide is fused to a nucleic acid sequence encoding a viral coat protein, eg, gene III protein or gene VIII protein. Monovalent phage display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused with a nucleic acid sequence encoding a portion of a gene III protein (Bass, S., Proteins, 8: 309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3: 205 (1991)). In a monovalent phage display system, the gene fusion is expressed at a low level and the wild type gene III protein is also expressed, so that the infectivity of the particles is retained. Methods for generating peptide libraries and screening those libraries have been disclosed in numerous patents (eg, US Pat. No. 5,723,286; US Pat. No. 5,432,018; US Pat. No. 5,580,717; US Pat. No. 5,427,908). And US Pat. No. 5,498,530).

  In some embodiments, Formula I, Formula II, or Formula III is expressed as a peptide library on phage. Phage expressing a library of polypeptides of formula I, formula II or formula III are then subjected to selection based on BLyS binding. In some embodiments, the selection process involves binding a phage to biotinylated BLyS, which is then bound to a neutravidin plate. Phage bound to the plate via BLyS-biotin-neutravidin binding are recovered and propagated. In some embodiments, the phage is subjected to several selections. In some embodiments, the phage is incubated with BLyS-biotin and then non-biotinylated BLyS is added as a competitive binding agent.

Further guidance on the use of phage display in the context of the present invention is provided in the examples.
Polypeptide fused or conjugated with heterologous polypeptide

  Fc fusion protein molecules comprising the polypeptides of the invention are further contemplated for use in the methods herein. In some embodiments, the molecule comprises a fusion of a polypeptide of the invention with an immunoglobulin or a specific region of an immunoglobulin. With respect to the bivalent form of the Fc fusion protein, such a fusion usually comprises the Fc region of an IgG molecule. In a further embodiment, the Fc region is from a human IgG1 molecule. In some embodiments, the immunoglobulin fusion includes the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of an IgG1 molecule.

  See also US Pat. No. 5,428,130, US Pat. No. 5,843,725, US Pat. No. 6,018,026 and Chamow et al., TIBTECH, 14: 52-60 (1996) for the production of immunoglobulin fusions.

  The simplest and most direct Fc fusion protein design often combines the binding domain (s), preferably the native sequence, of the antagonist polypeptides of the invention with the Fc region of an immunoglobulin heavy chain. In another embodiment, the polypeptide is artificial, eg, Formula I, Formula II, Formula III, ECFDLLVRAWVPCSVLK (SEQ ID NO: 13), ECFDLLVRHWVPCGLLR (SEQ ID NO: 14), ECFDLLVRRWVPCEMLG (SEQ ID NO: 15), ECFDLLVRSWVVPHMLR (SEQ ID NO: 15) 16) can be a polypeptide comprising the sequence of ECFDLLVRHWVACGLLR (SEQ ID NO: 17), and the sequence listed in FIG. 32 can be covalently linked to the Fc portion of an immunoglobulin. Furthermore, one or more of these polypeptides can be linked together and linked to the Fc portion of an immunoglobulin.

  Normally, when preparing the Fc fusion proteins of the invention, the nucleic acid encoding the binding domain is fused at the C-terminus with the nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however, N-terminal fusion is also possible. It is.

  Typically, in such a fusion, the encoded chimeric polypeptide retains at least a functionally active hinge, CH2 and CH3 domain of the constant region of an immunoglobulin heavy chain. The fusion is also made directly to the C-terminus of the Fc portion of the constant domain or directly to the N-terminus to the corresponding region of the heavy chain CH1 or light chain. The exact site at which the fusion is made is not critical; the particular site is well known and can be selected to optimize the biological activity, secretion or binding characteristics of the Fc fusion protein.

  In a preferred embodiment, the binding domain sequence is fused to the N-terminus of the Fc region of immunoglobulin G1 (IgG1). It is possible to fuse the complete heavy chain constant region to the binding domain sequence. More preferably, however, the sequence begins at the hinge region immediately upstream of the papain cleavage site that chemically limits IgG Fc (ie, residue 216, the heavy chain constant region is considered 114) or a similar site in other immunoglobulins. Used for fusion. In particularly preferred embodiments, the binding domain amino acid sequence is fused to (a) the hinge region and CH2 and CH3, or (b) the CH1, hinge, CH2 and CH3 domains of the IgG heavy chain.

  With respect to bispecific Fc fusion proteins, Fc fusion proteins are assembled as multimers, particularly as heterodimers or heterotetramers. In general, these aggregate immunoglobulins have a known unit structure. The basic 4-chain structural unit is the form in which IgG, IgD and IgE are present. Four chain units are repeated in high molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units held together by disulfide bonds. IgA globulins, and sometimes IgG globulins, can also exist in multimeric form in serum. In the case of multimers, each of the four units can be the same or different.

  Various exemplary assembled Fc fusion proteins within the scope herein are systematically illustrated as follows: (a) ACL-ACL; (b) ACH- (ACH, ACL-ACH, ACL -VHCH or VLCL-ACH); (c) ACL-ACH- (ACL-ACH, ACL-VHCH, VLCL-ACH or VLCL-VHCH); (d) ACL-VHCH- (ACH or ACL-VHCH or VLCL-ACH ); (E) VLCL-ACH- (ACL-VHCH or VLCL-ACH); and (f) (AY) n- (VLCL-VHCH) 2, where A is each a BLyS receptor Sequences derived from domains, sequences derived from antibodies to BLyS or to receptors for BLyS, or artificial sequences such as Formula I, Formula I , Formula III, ECFDLLVRAWVPCSVLK (SEQ ID NO: 5), ECFDLLVRHWVPCGLLR (SEQ ID NO: 6), ECFDLLVRRWVPCEMLG (SEQ ID NO: 7), ECFDLLVRSWVPCHMLLR (SEQ ID NO: 8), ECFDLLVRHWVACGLLR, SEQ ID NO: 8 Representing the same or different polypeptides comprising a combination of amino acid sequences;

  VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin long chain variable domain; CL is an immunoglobulin light chain constant domain; CH is an immunoglobulin heavy chain constant domain; A large integer; Y means the residue of a covalent crosslinker.

  For the sake of brevity, the structures show only important features; they do not show immunoglobulin linkages (J) or other domains, nor are the disulfide bonds shown. However, when such domains are required for binding activity, they are constructed to be in the normal position they occupy in an immunoglobulin molecule.

  Alternatively, the Fc sequence can be inserted between the immunoglobulin heavy and light chain sequences so that an immunoglobulin containing the chimeric heavy chain is obtained. In this embodiment, the Fc sequence is fused to the 3 'end of the immunoglobulin heavy chain in each arm of the immunoglobulin between the hinge and CH2 domains, or between the CH2 and CH3 domains. A similar construct has been reported by Hoogenboom et al., Mol. Immunol., 28: 1027-1037 (1991).

  The presence of an immunoglobulin light chain is not required in the Fc fusion proteins of the invention, but the immunoglobulin light chain is present covalently linked to a binding domain-immunoglobulin heavy chain fusion polypeptide, or It can be present fused directly to the binding domain. In the former case, the DNA encoding the immunoglobulin light chain is typically coexpressed with the DNA encoding the binding domain-immunoglobulin heavy chain fusion protein. Upon secretion, the hybrid heavy and light chains are covalently linked to provide an immunoglobulin-like structure comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs. Suitable methods for the preparation of such structures are disclosed, for example, in US Pat. No. 4,816,567 (issued March 28, 1989).

  Fc fusion proteins are most conveniently constructed by fusing a cDNA sequence encoding a binding domain in an open reading frame with an immunoglobulin cDNA sequence. However, fusion with genomic immunoglobulin fragments can also be used (see, eg, Aruffo et al., Cell, 61: 1303-1313 (1990); and Stamenkovic et al., Cell, 66: 1133-1144 (1991)). The latter type of fusion requires the presence of Ig regulatory sequences for expression. CDNA encoding the IgG heavy chain constant region can be isolated based on published sequences from DNA libraries derived from spleen or peripheral blood lymphocytes by hybridization or by polymerase chain reaction (PCR) techniques. The cDNA encoding the binding domain and immunoglobulin portion of the Fc fusion protein is inserted in tandem into a plasmid vector that directs efficient expression in a selected host cell.

  Certain modifications have been made to produce Fc sequences useful for making fusion molecules, such as BLyS antagonist Fc fusion molecules for use in the present invention. Specifically, to create Fc fusion proteins, six versions of modified human IgG1 Fc are generated, referred to as Fc-488, and Fc4, Fc5, Fc6, Fc7 and Fc8. Fc-488 (SEQ ID NO: 39) is intended for convenient cloning of a fusion protein containing a human γ1 Fc region, which was constructed using a wild type human immunoglobulin γ1 constant region as a template. Concerns about potential adverse effects due to unpaired cysteine residues have led to the decision to replace cysteines that normally disulfide bond with immunoglobulin light chain constant regions with serine residues. Additional changes were introduced in the codon encoding EU index position 218 to introduce a BgIII restriction enzyme recognition site to facilitate future DNA manipulation. These changes were introduced into the PCR product encoded on the PCR primers. Codons for EU index positions 216 and 217 were incorporated into the fusion protein partner sequence for the position of BgIII and to complete the Fc hinge region.

  Fc4, Fc5, and Fc6 contain mutations to reduce Fc-mediated effector function by reducing FcγRI binding and complete C1q binding. Fc4 contains the same amino acid substitution introduced in Fc-488. Additional amino acid substitutions were introduced to reduce potential Fc-mediated effector functions. Specifically, three amino acid substitutions were introduced to reduce FcγRI binding. These are substitutions at EU index positions 234, 235 and 237. Substitution at these positions has been shown to reduce binding to FcγRI (Duncan et al., Nature 332: 563 (1988)). These amino acid substitutions can also reduce FcγRIIa binding as well as FcγRIII binding (Sondermann et al., Nature 406: 267 (2000); Wines et al., J. Immunol. 164: 5313 (2000)).

Several groups have described the association of EU index positions 330 and 331 (amino acid residues 134 and 135 of SEQ ID NO: 6) with complement complement binding in complement C1q (Canfield and Morrison, J Exp. Med. 173: 1483 (1991); Tao et al., J. Exp. Med. 178: 661 (1993)). Amino acid substitutions at these positions were introduced into Fc4 to reduce complement binding. The C _H3 domain of Fc4 is identical to that found in the corresponding wild-type polypeptide, except that the stop codon is a potential dam methylation when the cloned DNA is grown in a dam + strain of E. coli. To eliminate the site, it was changed from TGA to TAA.

  In Fc5, the arginine residue at EU index position 218 was mutated back to lysine because the BgIII cloning scheme was not used in fusion proteins containing this particular Fc. The remaining Fc5 sequences are the same as described above for Fc4.

  Fc6 is identical to Fc5 except that the carboxyl terminal lysine codon is excluded. The C-terminal lysine of mature immunoglobulin is often removed from the mature immunoglobulin after translation and then secreted from B cells or removed into the serum circulation. As a result, C-terminal lysine residues are typically not found on circulating antibodies. As with Fc4 and Fc5 above, the stop codon in the Fc6 sequence was changed to TAA.

Fc7 is identical to wild type γ1 Fc except for the amino acid substitution at EU index position 297 located in the C _H2 domain. EU index position Asn-297 is the site of N-linked carbohydrate attachment. N-linked carbohydrate introduces a potential source of mutability in the recombinantly expressed protein due to potential batch-to-batch variations in the carbohydrate structure. In an attempt to eliminate this potential mutability, Asn-297 was mutated to a glutamine residue to prevent N-linked carbohydrate attachment at that residue position. The carbohydrate at residue 297 is also involved in Fc binding to FcRIII (Sondermann et al., Nature 406: 267 (2000)). Thus, removal of carbohydrate should generally reduce binding of recombinant Fc7 containing the fusion protein to FcγR. As above, the stop codon in the Fc7 sequence was mutated to TAA.

  Fc8 is identical to the wild-type immunoglobulin γ1 region shown in SEQ ID NO: 6, except that the cysteine residue at EU index position 220 is replaced with a serine residue. This mutation eliminated a cysteine residue that normally disulfide bonds with the immunoglobulin light chain constant region. The use of any of these specific Fc domains for the formation of BLyS antagonists is within the scope of the invention.

  These molecules in leucine zipper form are also contemplated by the present invention. A “leucine zipper” is a leucine-rich sequence that enhances, promotes, or drives dimerization or trimerization of its fusion partner (eg, the sequence or molecule to which the leucine zipper is fused or linked). Is a term in the art used to refer to. Various leucine zipper polypeptides have been described in the art. See, for example, Landschulz et al., Science, 240: 1759 (1988); US Pat. No. 5,716,805; WO 94/10308; Hoppe et al., FEBS Letters, 344: 1991 (1994); Maniatis et al., Nature, 341 : See 24 (1989). Those skilled in the art will appreciate that leucine zipper sequences can be fused at the 5 'or 3' end of the polypeptides of the invention.

  The polypeptides of the invention can also be modified to form a chimeric molecule by fusing the polypeptide with another heterologous polypeptide or amino acid sequence.

  According to some embodiments, such heterologous polypeptide or amino acid sequence is one that acts to oligomerize the chimeric molecule. In some embodiments, such chimeric molecules comprise a fusion of the polypeptide with a tag polypeptide that provides an epitope to which the tag antibody can selectively bind. The epitope tag is generally placed at the amino or carboxyl terminus of the polypeptide.

  The presence of such an epitope-tagged form of the polypeptide can be detected using an antibody against the tag polypeptide. Furthermore, providing an epitope tag allows the polypeptide to be readily purified by affinity purification using a tag antibody or another type of affinity matrix that binds to the epitope tag.

Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tag; flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8 2159-2165 (1988)]; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)]; Herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides include Flag peptide [Hopp et al., BioTechnology, 6: 1204-1210 (1988)]; KT3 epitope peptide [Martin et al., Science, 255: 192-194 (1992)]; "-Tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)]; and T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci: USA, 87: 6393-6397 (1990)].
Construction of peptide-polymer conjugates

  In some embodiments, a strategy for conjugation of a polymer of a synthetic peptide (eg, PEGylation) creates a special functionality that is reactive with each other by forming a conjugate bond in solution. Each consists of combining the peptides and PEG moieties possessed. Peptides can be readily prepared by conventional solid phase synthesis. The peptide is “preactivated” with an appropriate functional group at a specific site. The precursor is purified and fully characterized before reacting with the PEG moiety. Peptide ligation with PEG usually occurs in the aqueous phase and can be easily monitored by reverse phase analytical HPLC. PEGylated peptides can be readily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.

  In some embodiments, the peptide is covalently attached to the terminal reactive group on the polymer via one or more of the amino acid residues of the peptide, primarily by reaction conditions, polymer molecular weight, and the like. Polymers having reactive group (s) are contemplated herein as activated polymers. Reactive groups selectively react with free amino or other reactive groups on the peptide. Potential reactive sites include: the N-terminal amino group, the ε amino group on the lysine residue, and other amino, imino, carboxyl, sulfhydryl, hydroxyl and other hydrophilic groups. However, the type and amount of reactive groups selected for optimal results, and the type of polymer used to avoid having reactive groups that react with too many particularly active groups on the peptide. It is understood that it depends on the particular peptide used. In some embodiments, reactive residues (eg, lysine (K), modified unnatural amino acids, or other small molecules) can be substituted at a position suitable for conjugation.

  In some embodiments, the peptide comprises Formula I, Formula II, Formula III, ECFDLLVRAWVPCSVLK (SEQ ID NO: 5), ECFDLLVRHWVPCGLLR (SEQ ID NO: 6), ECFDLLVRRWVPCEMLG (SEQ ID NO: 7), ECFDLLVRSWVPCHMLR (SEQ ID NO: VLGHRL (SEQ ID NO: 8) No. 9) or the sequence listed in FIG. 32 of WO 05/000351 has a terminal reactive group.

  In some embodiments, the peptide comprises Formula I, Formula II, Formula III, ECFDLLVRAWVPCSVLK (SEQ ID NO: 5), ECFDLLVRHWVPCGLLR (SEQ ID NO: 6), ECFDLLVRRWVPCEMLG (SEQ ID NO: 7), ECFDLLVRSWVPCHMLR (SEQ ID NO: VLGHRL (SEQ ID NO: 8) No. 9) or at least one or more, preferably more than one of the polypeptides comprising the sequence listed in FIG. 32 of WO 05/000351. Polypeptides linked together may have the same sequence or have different sequences and terminal reactive groups. In some embodiments, these polypeptides can be tethered together, optionally through the use of a linker.

  Although conjugation can occur with any reactive amino acid on the polypeptide, in some embodiments, the reactive amino acid is linked to a reactive group of the activated polymer through its free ε-amino group. ), Or glutamic acid or aspartic acid, which is linked to the polymer via an amide bond. In some embodiments, the reactive amino acid of the peptide is not a cysteine residue at positions X2 and X1z.

  The degree of polymer conjugation with each peptide depends on the number of reactive sites on the polypeptide, the molecular weight of the polymer, the hydrophilicity and other attributes, and the particular peptide derivatization site selected. In some embodiments, the conjugate has a final molar ratio of 1-10 polymer molecules per peptide molecule, but more polymer molecules attached to the peptides of the invention are also contemplated. In some embodiments, each conjugate contains one polymer molecule. An experimental matrix in which the time, temperature and other reaction conditions are changed to vary the degree of substitution and then the level of polymer substitution of the conjugate is determined by size exclusion chromatography or other means known in the art By using the desired amount of derivatization is easily achieved.

  In some embodiments, the polymer contains only a single group that is reactive. This helps to avoid cross-linking of protein molecules. However, maximizing reaction conditions to reduce cross-linking or to purify reaction products by gel filtration or ion exchange chromatography to recover substantially homogeneous derivatives is within the scope herein. Is within. In other embodiments, the polymer contains two or more reactive groups for the purpose of linking multiple peptides to the polymer backbone.

  Furthermore, gel filtration or ion exchange chromatography can be used to recover the desired derivative in a substantially homogeneous form. In some embodiments, the polymer is covalently linked directly to the peptide without the use of a multifunctional (usually bifunctional) crosslinker. In some embodiments, the peptide chain to peptide molar ratio is 1: 1.

  A covalent modification reaction is used to react a biologically active substance with an inert polymer, preferably at about pH 5-9, more preferably 7-9, when the reactive group on the peptide is a lysine group. It can occur by any suitable method commonly used. In general, the process involves preparing an activated polymer (the polymer typically has at least one terminal hydroxyl group to be activated), preparing an active substrate from the polymer, and Thereafter, the peptide is reacted with an active substrate to produce a peptide suitable for the formulation. The modification reaction described above can be performed by several methods that can include one or more steps. Examples of modifiers that can be used to produce activated polymers in a one-step reaction include cyanuric chloride (2,4,6-trichloro-S-triazine) and cyanuric fluoride.

  In some embodiments, the modification reaction occurs in two steps, in which case the polymer first reacts with an acid anhydride, such as succinic anhydride or glutaric anhydride to produce a carboxylic acid, and then the carboxylic acid is Reacting with a compound capable of reacting with a carboxylic acid to produce an activated polymer having a reactive ester group capable of reacting with a peptide. Examples of such compounds include N-hydroxysuccinimide, 4-hydroxy-3-nitrile benzene sulfonic acid and the like, preferably N-hydroxy succinimide or 4-hydroxy-3-nitrobenzene sulfonic acid is used. For example, monomethyl substituted PEG can be reacted with glutaric anhydride at elevated temperature, preferably at about 100-110 ° C. for 4 hours. The methyl PEG-glutaric acid thus produced is then reacted with N-hydroxysuccinimide in the presence of a carbodiimide reagent, such as cyclohexyl or isopropylcarbodiimide, to activate the activated polymer methoxypolyethyleneglycolyl-N-succinate. This produces sinimidyl glutarate, which can then react with GH. This method is described in detail in Abuchowski et al., Cancer Biochem. Biophys., 7: 175-186 (1984). In another example, a monomethyl substituted PEG is reacted with glutaric anhydride and then with 4-hydroxy-3-nitrobenzenesulfonic acid (HNSA) in the presence of dicyclohexylcarbodiimide to produce an activated polymer. HNSA is described in Bhatnagar et al., Peptides: Synthesis-Structure-Func-tion. Proceedings of the Seventh American Peptide Symposium, Rich et al. (Eds.) (Pierce Chemical Co., Rockford III., 1981), p. 97. -100, and Nitecki et al., High-Technology Route to Virus Vaccines (American Society for Microbiology: 1986) entitled “Novel Agent for Coupling Synthetic Peptides to Carriers and Its Applications”.

  In some embodiments, the covalent bond with the amino group is accomplished by known chemistry based on cyanuric chloride, carbonyldiimidazole, aldehyde reactive groups (PEG alkoxide + diethyl acetal of bromoacetaldehyde; PEG + DMSO And acetic anhydride, or PEG chloride + 4-hydroxybenzaldehyde phenoxide, activated succinimidyl ester, activated dithiocarbonate PEG, 2,4,5-trichlorophenyl chloroformate or P-nitrophenyl chloroformate activation PEG). The carboxyl group is derivatized by coupling PEG-amine with carbodiimide. The sulfhydryl group is a maleimide-substituted PEG (for example, alkoxy-PEGamine + sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (described in WO97 / 10847 (published on March 27, 1997)) ), Or PEG-maleimide (commercially available from Nektar Technologies, San Carlos, CA (formerly Shearwater Polymers, Inc.)). Alternatively, free amino groups on the peptide (eg, ε-amino groups on lysine residues) can be coupled with N-hydroxysuccinimidyl substituted PEG (PEG-NHS available from Nektar Technologies). Or a maleimide-containing derivative of PEG as described in Pedley et al., Br. J. Cancer, 70: 1126-1130 (1994), or thiolated with 2-imino-thiolane (Trout reagent) Can be coupled with.

  A number of inert polymers such as, but not limited to, PEG are suitable for pharmaceutical use (eg, Davis et al., Biomedical Polymers: Polymeric Materials and Pharmaceuticals for Biomedical Use, pp.441-451 (1980)). reference). In some embodiments of the invention, non-proteinaceous polymers are used. Non-proteinaceous polymers are typically hydrophilic synthetic polymers, ie, polymers that would otherwise not be found in nature. However, polymers that exist in nature and are produced by recombinant or in vitro methods are useful, as are polymers isolated from native sources. Hydrophilic polyvinyl polymers such as polyvinyl alcohol and polyvinyl pyrrolidone are within the scope of the present invention. Particularly useful are polyalkylene ethers such as polyethylene glycol (PEG); polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (pluronic); polymethacrylates; carbomers; Dimeric D-mannose, D- and L-galactose, fucose, non-lactose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-glucose and neuraminic acid such as homopolysaccharide and Heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrin, glycogen, or polysaccharide subs of acid mucopolysaccharide Knit, such as hyaluronic acid; polymers of sugar alcohols, such as poly and polymannitol; heparin or Heparon.

  The polymer need not be water soluble prior to conjugation, but is preferably water soluble and the final conjugate is preferably water soluble. Preferably, the conjugate exhibits a water solubility of at least about 0.01 mg / ml, more preferably at least about 0.1 mg / ml, more preferably at least about 1 mg / ml.

  Further, the polymer should not be highly immunogenic in the conjugate form, or is incompatible with intravenous infusion, injection or inhalation when the conjugate is intended to be administered by routes such as intravenous or inhalation. It should not have a viscosity of.

  The molecular weight of the polymer can range up to about 100,000 D, and is preferably at least about 500 D, or at least about 1,000 D, or at least about 5,000 D. In some embodiments, PEG or other polymer has a molecular weight in the range of 5000-20,000 D. The molecular weight chosen depends on the effective size of the conjugate to be achieved, the nature of the polymer (eg structure, eg linear or branched) and the degree of derivatization, ie the number of polymer molecules per peptide, and the poly It may depend on the polymer attachment site (s) on the peptide. In some embodiments, branched PEG can be used to induce a large increase in the effective size of the peptide. PEG or other polymer conjugates can be utilized to increase half-life, increase solubility, stabilize against proteolytic attack, and reduce immunogenicity.

  Functionalized PEG polymers for modifying the peptides of the present invention are available from Nektar Technologies, San Carlos, CA (formerly Shearwater Polymers, Inc.). Such commercially available PEG derivatives include amino-PEG, PEG amino acid ester, PEG-N-hydroxysuccinamide chemistry (NHS), PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG, PEG-propion. Acid, PEG amino acid, PEG succinimidyl succinate, PEG succinimidyl propionate, succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidyl ester of amino acid PEG, PEG -Oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether, PEG-anhydride, PEG vinyl sulfone, PEG-maleimide, PEG-orthopyridyl-disulfide, Hetero-functional PEG, PEG vinyl derivatives, PEG silanes, and PEG phospholides include, but are not limited to. Reaction conditions for coupling these PEG derivatives vary depending on the protein, the desired degree of PEGylation, and the PEG derivative utilized. Some factors involved in the selection of PEG derivatives include the following: desired attachment point (eg, lysine or cysteine R-group), hydrolytic stability and reactivity of the derivative, stability of the linkage, Toxicity and antigenicity, analytical suitability, etc. Specific instructions for use of any particular derivative are available from the manufacturer.

  Conjugates can be characterized by SDS-PAGE, gel filtration, NMR, trypsin mapping, liquid chromatography-mass spectrometry, and in vitro bioassays. For example, the degree of PEG conjugation can be shown by SDS-PAGE and gel filtration, and then analyzed by NMR, which has a specific resonance peak for the methylene hydrogen of PEG. The number of PEG groups on each molecule can be calculated from NMR spectra or mass spectroscopy. Polyacrylamide gel electrophoresis in 10% SDS is suitably performed in 10 mM Tris-HCl, pH 8.0, 100 mM NaCl as the elution buffer. Trypsin mapping can be performed to demonstrate which residues are PEGylated. Thus, PEGylated peptides are trypsin in 100 mM sodium acetate, 10 mM Tris-HCl, 1 mM calcium chloride, pH 8.3, at 37 ° C. for 4 hours at a protein / enzyme ratio of 100 to 1 (in mg). After digestion and oxidation to pH <4, the digestion is stopped and then separated on HPLC Nucleosil C-18 (4.6 mm..150 mm, 5. mu, 100A). The chromatogram is compared to the chromatogram of the non-PEGylated starting material. Each peak can then be analyzed by mass spectrometry to verify the size of the fragment in the peak. Fragment (s) with PEG groups are usually not retained on the HPLC cam after injection and disappear from the chromatogram. Such disappearance from the chromatogram is an indication of PEGylation for a particular fragment that should contain at least one lysine residue. The PEGylated peptide can then be assayed for the ability to bind to BLyS by conventional methods.

  In some embodiments, the conjugate is purified by ion exchange chromatography (eg, ion exchange HPLC). Many chemistries of electrophilically activated PEG cause a reduction in the amino group charge of the PEGylated product. Thus, high resolution ion exchange chromatography can be used to separate free and conjugated proteins and resolve species that exhibit different levels of PEGylation.

  Indeed, resolution of different species (eg containing 1 or 2 PEG residues) is also possible due to differences in the ionic properties of the unreacted amino acids. In one embodiment, species exhibiting different levels of PEGylation are resolved according to the method described in WO 96/34015 (International Application PCT / US96 / 05550 (published Oct. 31, 1996)). Heterogeneous conjugates are purified from each other in the same manner.

  In some embodiments, PEG-N-hydroxysuccinamide (NHS) reacts with primary amines (eg, lysine and the N-terminus). In some embodiments, PEG-NHS reacts with the C-terminal lysine (K) of the polypeptide. In some embodiments, a lysine residue is added to the C-terminus of the 17-mer polypeptide, while in other embodiments, Xi is replaced with lysine. In some embodiments, the polymer reacts with the N-terminus. In preferred embodiments, conjugates are generated utilizing the derivatization and purification methods described in the examples below.

  In one embodiment, the present invention provides any of the conjugates described above, i.e. one or more polymers, formed by its constituent parts without any extraneous material in the covalent molecular structure of the conjugate. One or more peptide (s) covalently attached to the molecule (s) are provided.

  The production methods and products of the invention use or incorporate antibodies that bind to BLyS or one or more of its three receptors. Accordingly, methods for generating such antibodies are described herein.

  The BLyS or BLyS receptor to be used for the production of antibodies or for screening for antibodies can be, for example, a soluble form of the antigen or a part thereof containing the desired epitope. As noted above, BLyS sequences and BLyS receptor sequences are known as the boundaries of the various domains of these polypeptides. Peptide fragments of the extracellular domain (ECD) can be used as immunogens. Based on a summary description of these known sequences and domains, one of skill in the art can express BLyS or BLyS receptor polypeptides and fragments thereof for use in producing antibodies.

  To generate antibodies against BLyS or its receptor, full-length polypeptides, or peptide fragments of 6 or more residues in length, are used as immunogens in rabbits such as mice, hamsters and rats, and rabbits Antibodies can be produced in goats or other suitable animals. A positive BLyS or BLyS receptor polypeptide or immunogenic fragment thereof can be expressed in a suitable host cell, eg, a bacterial or eukaryotic cell. In one embodiment, human and murine detergent solubilized full-length BLyS is produced in E. coli and used to immunize and screen for hybridomas producing BLyS antibodies.

  Alternatively, or additionally, B cells or cell lines that express BLyS or BLyS receptors on their cell surface can be used to generate and / or screen for antibodies. Other forms of BLyS useful for generating antibodies will be apparent to those skilled in the art and phage display methods can also be used to produce BLyS binding antibodies. An antibody that binds a BLyS or BLyS receptor can be a chimeric, humanized or human antibody. Such antibodies and methods for their production are described in further detail below.

  Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Bifunctional or derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (conjugated via cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, SOC12 or R1N = C = Proteins that are immunogenic in the species to be immunized using NR (wherein R and R ′ are different alkyl groups), such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibition It may be useful to conjugate the agent with the relevant antigen.

  For example, by injecting 100 wu or 5 g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution subcutaneously at multiple sites, the animal becomes antigen, immunogenic conjugate Immunized against the body or derivative. One month later, animals are boosted with the peptide or conjugate in 1/5 to 1/10 of the original amount of Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer reaches a plateau. Preferably, the animals are the same antigen but are conjugated to different proteins and / or boosted with conjugates via different crosslinkers. Conjugates can also be made in recombinant cell culture as protein fusions. Furthermore, aggregating agents, such as alum, are suitably used to enhance the immune response.

  Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are the same, provided that possible natural mutations may be present in small amounts. Thus, the modifier “monoclonal” refers to the nature of the antibody when it is not a mixture of different antibodies.

  For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or made by recombinant DNA methods (US Pat. No. 4,816,567). obtain. In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described hereinabove to produce antibodies that specifically bind to the protein used for immunization. Draw lymphocytes that can either be produced. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using an appropriate fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). )).

  The hybridoma cells thus prepared are seeded and grown in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the medium for the hybridoma typically comprises hypoxanthine, aminopterin and thymidine (HAT medium) This substance prevents the growth of HGPRT-deficient cells.

  Preferred myeloma cells fuse efficiently, support stable high level production of antibodies by selected antibody producing cells, and are sensitive to a medium such as HAT medium.

  Of these, preferred myeloma cell lines are murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, California USA), and SP- 2 or X63-Ag8-653 cells (available from American Type Culture Collection, Rockville, Maryland USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

  Medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). For example, the binding affinity of a monoclonal antibody can be determined by Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).

  After hybridoma cells producing antibodies with the desired specificity, affinity and / or activity have been identified, clones can be subcloned by limiting dilution techniques and grown by standard methods (Goding, Monoclonal Antibodies Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, hybridoma cells can be grown in vivo as ascites tumors in animals.

  Monoclonal antibodies secreted by subclones are suitably separated from the medium, ascites or serum by conventional immunoglobulin production techniques such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. The The DNA encoding the monoclonal antibody is readily isolated using conventional techniques (eg, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of murine antibodies). , Sequenced. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is placed in an expression vector which, in turn, is a host cell that does not otherwise produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or It can be transfected into myeloma cells to synthesize monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol Revs., 130 : 151-188 (1992).

  In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990).

  Clackson et al., Nature, 352: 624-628 (1991) and Marks etal., J. Mol. Biol., 222: 581-597 (1991) are the respective antibodies for murine and human antibodies using phage libraries. The isolation of is described. Subsequent publications produced high affinity (nM range) human antibodies by chain shuffling (Marks et al., BiolZ'echraology, 10: 779-783 (1992)), as well as construction of very large phage libraries Combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)) have been described as strategies for achieving this. Thus, these techniques can be performed in place of traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

  DNA is substituted, for example, by coding sequences for human heavy and light chain constant domains in place of homologous murine sequences (US Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81 6851 (1984)), or can also be modified by covalently linking all or part of the coding sequence for the non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

  Typically, such non-immunoglobulin polypeptides are substituted for the constant domain of an antibody, or they are substituted for the variable domain of one antigen-binding site of an antibody to determine specificity for the antigen. A chimeric bivalent antibody is produced which comprises another antigen-binding site having specificity for a different antigen from a convenient site under one antigen.

  Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is essentially the method of Winter and his collaborators (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)) can be performed by replacing the hypervariable region sequence with the corresponding sequence of the human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies in which a substantially variable human variable domain is replaced by the corresponding sequence from a non-human species (US Pat. No. 4,816,567). In fact, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

  The selection of human variable domains (both light and heavy chains) to be used for the production of humanized antibodies is very important to reduce antigenicity. According to the so-called “best fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human framework region (FR) for humanized antibodies (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al. al., J. Mol. Biol., 196: 901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light and heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol. 15-1: 2623 (1993)).

  It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies are prepared by a process of analysis of the parent sequence and various conceptual humanized products, using a three-dimensional model of the parent and humanized sequences, according to a preferred method.

  Three-dimensional immunoglobulin models are commercially available and are well known to those skilled in the art. Computer programs are available that describe and display the predicted three-dimensional conformational structure of selected candidate immunoglobulin sequences. By examining these representations, it is possible to analyze the role of residues in the function of a candidate immunoglobulin sequence, ie, to analyze residues that affect the ability of a candidate immunoglobulin to bind its antigen. . In this way, FR residues are selected and combined from the recipient and import sequences, thus achieving increased affinity for the desired antibody characteristic, eg, target antigen (s). In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

  As an alternative to humanization, human antibodies can be generated. For example, it is now possible to create a transgenic animal (eg, a mouse) that can produce a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutated mice results in complete suppression of endogenous antibody production.

  Transfer of the human germline immunoglobulin gene in such germline mutant mice causes production of human antibodies upon antigen challenge. For example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369, and 5,545,807.

  Alternatively, human antibodies can be generated in vitro from immunoglobulin variable (V) domain gene repertoires from non-immunized donors using phage display techniques (McCafferty et al., Nature 348: 552-553 (1990)). And antibody fragments can be produced. According to this technique, antibody V domain genes are cloned in reading frames in filamentous bacteriophages, such as large or small coat protein genes of M13 or fd, and displayed as functional antibody fragments on the surface of phage particles. Since filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also selects for a gene encoding an antibody exhibiting that property. Thus, phage mimics some of the characteristics of B cells.

  Phage display can be performed in a variety of formats: see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993) for their review. . Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diversity array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from non-immunized human donors has been constructed and antibodies against a diversity array of antigens (eg, autoantigens) have been developed by Marks et al., J. Mol. Biol. 222: 581-597 (1991 ) Or Griffith et al., EMBO J. 12: 725-734 (1993). See also US Pat. Nos. 5,565,332 and 5,573,905. Human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

  Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab′-SH fragments can be recovered directly from E. coli and chemically combined to form F (ab ′) 2 fragments (Carter et al., Bio / Technology 10: 163-167). (1992)). According to another approach, F (ab ') 2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, specifically selected antibodies are single chain Fv fragments (scFv) (see WO 93/16185; US Pat. No. 5,571,894 and US Pat. No. 5,587,458). The antibody fragment may also be a “linear antibody” as described, for example, in US Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

  Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of a B cell surface marker. Other such antibodies may bind a first B cell marker and further a second B cell surface marker. Alternatively, the anti-B cell marker binding arm may be a triggering molecule on leukocytes, such as a T cell receptor molecule (eg, CD2 or CD3), or an Fc against IgG, to focus on cell defense mechanisms against B cells. It can be combined with an arm that binds to a receptor (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16). Bispecific antibodies can also be used to localize cytotoxic agents against B cells.

  These antibodies possess a B cell marker binding arm that binds to a cytotoxic agent (eg, saporin, anti-interferon, vinca alkaloid, ricin A chain, methotrexate or radioisotope hapten).

  Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ') bispecific antibodies). Bispecific antibody production methods are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature , 305: 537-539 (1983)).

  Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Accurate molecular purification (usually performed by affinity chromatography steps) is rather cumbersome and the product yield is low. Similar approaches are disclosed in WO 93/08829 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

  According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. The first heavy chain constant region (CH1) containing the site necessary for light chain binding is preferably present in at least one of the fusions. For immunoglobulin heavy chain fusions, if desired, the DNA encoding the immunoglobulin light chain is inserted into a separate expression vector and cotransfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where the unequal proportion of the three polypeptide chains used in the construction provides optimal yield. However, if expression of an equal ratio of at least two polypeptide chains results in high yields, or if the ratio has no special meaning, it relates to two or all three polypeptide chains in an expression vector. It is possible to insert a coding sequence.

  In a preferred embodiment of this approach, the bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (second binding in the other arm). Providing specificity). Asymmetric structure facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of the immunoglobulin light chain in only half of the bispecific molecule provides an easy method of separation It was found that This approach is disclosed in WO 94/04690. For further details on generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986). According to another approach described in US Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from the recombinant cell medium. A preferred interface comprises at least part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). A compensatory “cavity” of the same or similar size as the large side chain (s) replaces the large amino acid side chain with a smaller one (eg alanine or threonine) on the interface of the second antibody molecule. Produced. This provides a mechanism for increasing the yield of heterodimers over other undesirable end products such as homodimers.

  Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heterozygote can be bound to avidin and the other bound to biotin. Such antibodies target immune system cells, for example against unwanted cells (US Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Has been proposed for. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable crosslinkers are well known in the art and are disclosed in US Pat. No. 4,676,980 along with a number of crosslinking techniques.

  Techniques for the generation of bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a technique in which intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The produced Fab 'fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab′-TNB derivatives is then reconverted to Fab′-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of other Fab′-TNB derivatives to produce a bispecific Forming a sex antibody. The bispecific antibody produced can be used as an agent for selective immobilization of enzymes.

  Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli that can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describes the production of fully humanized bispecific antibody F (ab ') 2 molecules. Each Fab 'fragment was secreted separately from E. coli and subjected to directed chemical binding in vitro to form bispecific antibodies. The bispecific antibody thus formed bound to ErbB2 receptor overexpressing cells and normal human T cells and induced lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

  Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by gene fusion.

  The antibody homodimer was reduced at the hinge region to form a monomer and then reoxidized to form an antibody heterodimer. This method can also be utilized for the production of antibody homodimers.

  The “diabody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. Provided. The fragment includes a heavy chain variable domain (VH) that is linked to the light chain variable domain (VL) by a linker that is too short to allow pairing between two domains on the same chain. Thus, the VH and VL domains of one fragment are strongly paired with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for generating bispecific antibody fragments by the use of single chain Fv (sFv) dimers has also been reported (see Gruber et al., J. Immunol., 152: 5368 (1994)). ).

  Antibodies with a titer greater than 2 are contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60 (1991)).

  Amino acid sequence modification (s) of the protein or peptide antagonists and antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of a BLyS binding antibody or antagonist. Amino acid sequence variants of the antagonist are prepared by introducing appropriate nucleotide changes into the antagonist nucleic acid, or by peptide synthesis. Such modifications include, for example, the deletion of residues from the amino acid sequence of the antagonist and / or the insertion and / or substitution of residues within the amino acid sequence. Any combination of deletions, insertions and substitutions will arrive at the final construct, provided that the final construct retains the desired attributes. Amino acid changes can also alter the post-translational process of the antagonist, such as a change in the number or position of glycosylation sites.

  A useful method for the identification of certain residues or regions of an antagonist that is the preferred position for mutagenesis is described in “Alanine Scanning Suddenly, as described in Cunninghham and Wells Science, 244: 1081-1085 (1989). It is called “mutagenesis”. Here, a group of residues or target residues is identified (eg charged residues such as arg, asp, his, lys and glu) and taken by neutral or negatively charged amino acids (most preferably alanine or polyalanine). Instead, it affects the interaction between amino acids and antigens. Next, amino acid positions demonstrating functional sensitivity to the substitution are improved by introducing additional or other variants at or for the site of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the achievement of mutations at a given site, tala scanning or random mutagenesis is performed at the target codon or region and the expressed antagonist variants are screened for the desired activity.

  Amino acid sequence insertions are amino- and / or carboxyl-terminal fusions ranging in length from 1 residue to a polypeptide containing 100 or more residues, and within sequences of single or multiple amino acid residues Includes insertions. Examples of terminal insertions include antagonists having an N-terminal bud thionyl residue or antagonists fused to a cytotoxic polypeptide. Other insertional variants of the antagonist molecule include fusions with the N- or C-terminus of the enzyme's antagonist, or a polypeptide that increases the antagonist serum half-life.

  Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antagonist molecule that is replaced by a different residue. Sites that have the greatest benefit for substitutional mutagenesis of antibody antagonists include hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions”. If such a substitution results in a change in biological activity, a more substantial change is introduced as referred to in Table 1 as an “exemplary substitution” or as further described below for the amino acid class, The product is screened.

  Substantial modifications in the biological properties of the antagonist include (a) the structure of the polypeptide backbone in the region of substitution as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or ( c) It is achieved by selecting substitutions whose action on retaining most side chains is significantly different. Natural residues are divided into groups based on common side chain properties: (1) hydrophobicity: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilicity: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatics: trp, tyr, phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class.

  Any cysteine residue that is not involved in maintaining the proper conformation of the antagonist may generally be substituted with serine to improve the oxidative stability of the molecule and prevent ectopic cross-linking. Conversely, cysteine bond (s) can be added to an antagonist to improve its stability (particularly where the antagonist is an antibody fragment, eg, an Fv fragment).

  A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. In general, the resulting variant (s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way to generate such substitutional variants is affinity maturation using phage display. In short, some hypervariable region sites (eg 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as a fusion with the gene III product of M13 packaged within each particle. The phage display variants are then screened for their biological activity (eg, binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is subjected to screening as described herein to further develop antibodies with superior properties in one or more related assays. Can be selected for.

  Another type of amino acid variant of the antagonist alters the original glycosylation pattern of the antagonist. By altering is meant deleting one or more carbohydrate moieties found in the antagonist, and / or adding one or more glycosylation sites that are not present in the antagonist.

  Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic coupling of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the linkage of one of the sugars N-acetylgalactosamine, galactose or xylose with a hydroxy amino acid, most commonly near h-serine or threonine, but also 5-hydroxyproline or 5-hydroxylysine Can be used.

  Addition of a glycosylation site to the antagonist is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by addition of, or substitution by, one or more serine or threonine residues to the original antagonist sequence (with respect to the O-linked glycosylation site).

  Nucleic acid molecules encoding amino acid sequence variants of the antagonist are prepared by a variety of methods known in the art. These methods include isolation from natural sources (in the case of natural amino acid sequence variants), or oligonucleotide-mediated (or site-specific) mutagenesis, PCR mutagenesis and early preparation variants of antagonists or Examples include, but are not limited to, preparation by non-mutant versions of cassette mutagenesis.

  For example, it is desirable to modify the antagonists of the present invention with respect to effector function to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antagonist. This can be accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody antagonist. Alternatively or additionally, cysteine residue (s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.

  The homodimeric antibody thus generated may have improved internalization capabilities and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (Caron et al. al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, BJ Immunol. 148: 2918-2922 (1992)). Homodimeric antibodies exhibiting enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking agents as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, antibodies with a dual Fc region can be engineered to thereby have enhanced complement lysis and ADCC capabilities (Stevenson et al., Anti-Cancer Drug Design 3: 219-230 ( 1989)).

In order to increase the serum half-life of the antagonist, one may incorporate a salvage receptor binding epitope into the antagonist (especially an antibody fragment) as described in US Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to the Fc region of an IgG molecule (eg, IgG1, IgG2, IgG3 or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule. Refers to the epitope.
Test

  Peripheral B cell concentration is determined by the FACS method counting CD3- / CD40 + cells.

The percentage of CD3-CD40 + B cells out of total lymphocytes in the sample is obtained by the following gating strategy. The lymphocyte population is marked on the forward scatter / side scatter scattergram to limit region 1 (Ri). Using events in RI, fluorescence intensity dot plots are displayed for CD40 and CD3 markers. Fluorescently labeled isotype controls are used to establish the respective cutoff points for CD40 and CD3 positivity.
FACS analysis

  500,000 cells are washed and resuspended in 100 l FACS buffer (phosphate buffered saline with 1% BSA containing 5 ul of staining or control antibody). All stained antibodies, including isotype controls, are obtained from PharMingen, San Diego, CA. Human BLYS expression is assessed by staining with Rituxan & commat with a FITC-conjugated anti-human IgG1 secondary antibody.

  FACS analysis is performed using FACScan and Cell Quest (Becton Dickinson Immunocytometry Systems, San Jose, CA). All lymphocytes are limited in forward and side light scatter, while all B lymphocytes are limited in B220 expression on the cell surface.

Analysis of hBLYS + B cells by FACS in the spleen, lymph nodes and bone marrow, by analyzing peripheral B cell counts and on a daily basis for the first week after injection and weekly thereafter, reduces B cell depletion and recovery. Assessed. Serum levels of injected 2H7 variant antibody are monitored.
Formulation

A therapeutic formulation of a BLyS antagonist, such as a BLyS binding antibody used in accordance with the present invention, can be prepared in the form of a lyophilized formulation or an aqueous solution of an antibody having the desired degree of purity, any pharmaceutically acceptable carrier, excipient. It is prepared by mixing or stabilizer ^{(Remitgtorz's Pharmamaceutical Science 16 th edition} , Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are independent of the dosage used and non-toxic to the recipient, and buffers such as phosphates, citrates and other organic acids; oxidation Inhibitors, ascorbic acid and methionine; preservatives (eg octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol Resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as poly Vinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose Or a sorbitol; a salt-forming counterion, such as sodium; a metal complex (eg, a Zn-protein complex); and / or a non-ionic surfactant, such as Tween, Pluronic ™ or polyethylene glycol (PEG).

  The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it provides cytotoxic drugs, chemotherapeutic drugs, cytokines or immunosuppressive drugs (eg those that act on T cells, eg cyclosporine or antibodies that bind T cells, eg those that bind LFA-1) Is desirable. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used at the same dosage and route as described herein, or at about 1-99% of the dosage used so far.

The active ingredients are for example in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example in hydroxymethylcellulose or colloid drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules). Alternatively, they can be encapsulated in gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively, in a macroemulsion. Such ^{techniques, Remington's Pharmaceutical Sciences 16 th edition} , Osol, are disclosed in A. Ed. (1980).

  Sustained release preparations can be prepared. Suitable examples of sustained release preparations include solid hydrophobic polymer semipermeable matrices containing antagonists, which are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L-glutamic acid and ethyl-L-glutamate. Copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxy Examples include butyric acid.

The formulation to be used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes.
Disease treatment disease

  The anti-CD20 agents and BLyS antagonists of the present invention are useful for treating B cell regulatory autoimmune disorders. B cell-regulated autoimmune diseases include arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis such as atopic dermatitis, chronic autoimmune urticaria, polymyositis / skin Myositis, toxic epidermal necrosis, systemic scleroderma and sclerosis, responses related to inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis), respiratory distress syndrome, adult respiratory distress syndrome (ARDS) , Meningitis, allergic rhinitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic symptoms, eczema, asthma, symptoms with T cell infiltration and chronic inflammatory response, atherosclerosis, autoimmunity Myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), lupus (eg, nephritis, non-renal, discoid, alopecia), juvenile-onset diabetes, multiple sclerosis, allergic encephalomyelitis , Rhino Immune response associated with acute and delayed hypersensitivity mediated by Cain and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis such as Wegener's granulomatosis, granulocytopenia, vasculitis (eg ANCA), poor regeneration Anemia, Coombs-positive anemia, diamond blackfan anemia, immune hemolytic anemia such as autoimmune hemolytic anemia (AIHA), pernicious anemia, erythroblast dysplasia (PRCA), factor VIII deficiency, type A hemophilia Disease, autoimmune neutropenia, pancytopenia, leukopenia, disease with leukocyte leakage, CNS inflammatory disorder, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated disease, anti Glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture syndrome, Lambert -Tonton myasthenia syndrome, Raynaud's syndrome, Sjogren's syndrome, Steven Johnson syndrome, solid organ graft rejection (eg, high panel reactive antibody titer, IgA deposition in tissues, etc.), graft versus host disease (GVHD), Bullous pemphigoid, pemphigus (including vulgaris, deciduous), autoimmune multiglandular endocrine disorder, Reiter's disease, stiff man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, testicular and ovarian autoimmune diseases, Eg autoimmune testitis and ovitis, primary hypothyroidism; autoimmune endocrine diseases such as autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis) , Subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune multi-gland syndrome (or multi-gland endocrine disorder syndrome), type I diabetes (also called insulin-dependent diabetes mellitus (IDDM)) And Sheehan syndrome; autoimmune hepatitis, lymphoid interstitial pneumonia (HIV), obstructive bronchiolitis (non-graft) vs. NSIP, Guillain-Barre syndrome, large vasculitis (eg Kawasaki disease and nodular polyposis Arteritis), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, childhood steatosis (gluten enteropathy), cold globulinemia, ALS, coronary artery Disease.

  The desired level of B cell depletion depends on the disease. Preferably, B cell depletion is sufficient to prevent disease progression for at least 2 months, more preferably 3 months, more preferably 4 months, more preferably 5 months, more preferably 6 months or more. It is. In a more preferred embodiment, B cell depletion is at least 6 months, more preferably 9 months, more preferably 1 year, more preferably 2 years, more preferably 3 years, more preferably 5 years or more, in remission. Enough to increase the time. In the most preferred embodiment, B cell depletion is sufficient to cure the disease. In a preferred embodiment, B cells in autoimmune disease patients are at least about 75%, more preferably 80%, 85%, 90%, 95%, 99% of baseline levels prior to treatment, 100% Even%.

  For the treatment of autoimmune diseases, it is desirable to adjust the degree of B cell depletion depending on the severity of the disease and / or symptoms in an individual patient by adjusting the dose of an immunosuppressant or BLyS antagonist. Thus, B cell depletion can be complete, but not necessarily complete. Alternatively, total B cell depletion may be desired in the initial treatment, but in subsequent treatments the dosage may be adjusted to achieve only partial depletion. In one embodiment, B cell depletion remains at least 20%, ie, 80% or less B cells remain when compared to baseline levels prior to treatment. In other embodiments, B cell depletion is 25%, 30%, 40%, 50%, 60%, 70% or more.

  Preferably, B cell depletion is sufficient to stop disease progression, preferably to reduce the signs and symptoms of the particular disease being treated, more preferably to cure the disease .

  For therapeutic applications, the immunosuppressant and BLyS antagonist compositions of the present invention can be used in combination therapy with additional agents such as anti-inflammatory agents such as DMARDS and other biologics. Prior therapy may be administered in conjunction with other forms of conventional therapy for autoimmune disease, either before or after conventional therapy.

  If there is a measurable improvement in symptoms or other applicable criteria after administration of the composition of the invention compared to before treatment, the method of the invention may cause patients with B cell-regulated autoimmune disease to Are alleviated and treated successfully. The effect of treatment can be evident within 3 to 10 weeks after administration of the composition of the invention. Applicable criteria for each disease are well known to skilled medical practitioners. For example, a physician can monitor a treated patient for clinical or serological evidence of disease, such as serological markers of disease, complete blood counts, such as B cell counts, and serum immunoglobulin levels. Serum levels of IgG and IgM are reduced in BLyS antagonists such as atacicept treated mice. Human patients who respond to atacicept treatment similarly show a reduction in serum IgG and IgM levels.

  Parameters for assessing the efficacy and success of treatment of autoimmunity or autoimmune related diseases are known to skilled practitioners in matched diseases. In general, skilled physicians look for signs of specific diseases and reduction of symptoms. An example is shown below.

  Rheumatoid arthritis (RA) is an autoimmune disorder of unknown etiology. Most RA patients have a chronic course of disease that can cause progressive joint destruction, deformity, disability, and even premature death, even when using treatment. The goal of both RA is to prevent or control joint damage, prevent loss of function, and reduce pain (“Guidelines for the management of rheumatoid arthritis” Arthritis & Rhemmatism 46 (2): 328- 346 (February, 2002)). The vast majority of patients with newly diagnosed RA are started with disease modifying anti-rheumatic drug (DMARD) therapy within 3 months of diagnosis.

  DMARDs commonly used for RA are hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (+ oral and subcutaneous methotrexate), azathioprine, D-penicillamine, gold (oral), gold (intramuscular), minocycline, cyclosporine, grape Coccus protein A immunoadsorption.

  Since the body produces tumor necrosis factor α (TNFa) in RA, TNFa inhibitors have been used to treat the disease. Etanercept (ENBREL) is an injectable drug approved in the United States for the treatment of active RA. Etanercept binds to TNFa and removes most of the TNFa from the joints and blood, thereby preventing TNFa from promoting inflammation and other symptoms of rheumatoid arthritis.

  Etanercept is a “Fc fusion protein” fusion protein consisting of the extracellular ligand binding portion of human 75 kD (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1.

  Infliximab (sold under the brand name Remicade) is an immunosuppressive drug prescribed to treat RA and Crohn's disease. Infliximab is a chimeric monoclonal antibody that targets TNFα that binds to TNF and produces inflammation, thereby reducing inflammation in the body by binding to it.

Adalimumab (Humila ^™ , Abbott Laboratories), formerly known as D2E7, binds to TNFa and has moderate to severely active RA that has had an inadequate response to one or more traditional disease modifying DMARDs. Approved to reduce signs and symptoms in adults and to suppress the progression of structural damage.

  Treatment of rheumatoid arthritis by administering an immunosuppressive drug and a BLyS antagonist can be performed in conjunction with treatment with one or more of the drugs for RA. For example, for rheumatoid arthritis, measures related to treatment progression may include the number of swollen and tender joints, and the length of morning stiffness. The patient can be examined how many joints in the hand and foot are being eroded using a scoring system known as X-ray and sharp score. Another scoring system is based on the American College of Rheumatology criteria for assessing response to therapy.

One method of assessing therapeutic efficacy in RA is based on the American College of Rheumatology (ACR) criteria, which measure, among other things, the percentage improvement in tenderness and swollen joints. RA patients can be scored with an ACR20 (20% improvement), for example, without antibody treatment (eg baseline before treatment) or when compared to placebo treatment. Another method of assessing the efficacy of antibody therapy includes X-ray scoring, such as sharp X-ray scores used to score structural damage, such as osteotomy and joint gap narrowing. Patients may also be evaluated for prevention or improvement of disability based on health status questionnaire [HAQ] score, AIM score, SF distribution 36 during or after treatment. ACR20 criteria may include a 20% improvement in both tender (painful) joint number and swollen joint number plus at least 3 20% improvement in the following 5 additional measures:
1. Patient pain assessment by visual analog scale (VAS),
2. The patient's overall assessment for disease activity (VAS),
3. The physician's overall assessment of disease activity (VAS),
4). Impaired self-assessment ability of patients as measured by health questionnaire
5. Acute phase reactant, CRP or ESR.

  ACR50 and 70 are defined similarly. Preferably, the patient has an amount of BLyS binding antibody of the invention effective to achieve a score of at least ACR20, preferably at least ACR30, more preferably at least ACR50, more preferably at least ACR70, most preferably at least ACR75 and above. Is administered.

  Psoriatic arthritis has unique and different radiographic features. For psoriatic arthritis, joint erosion and joint gap narrowing can be similarly assessed by a sharp score. The humanized BLYS binding antibodies disclosed herein can be used to prevent joint damage and reduce disease signs and symptoms of the disorder.

  Yet another aspect of the invention is a method of treating lupus or SLE by administering a therapeutically effective amount of a BLyS antagonist in combination with an immunosuppressive drug of the invention to a patient suffering from SLE. The SLEDAI score provides a numerical quantification of disease activity. SLEDAI is a metric index of 24 clinical and laboratory parameters known to correlate with disease activity in a numerical range of 0 to 103 (Bryan Gescuk & John Davis, “Novel therapeutic agent for systemic lupus erythematosus ”in Current Opinion in Rheumatology 2002, 14: 515-521). Antibodies against double-stranded DNA are thought to cause lupus renal redness and other manifestations. Patients undergoing antibody treatment can be monitored for time to renal redness, defined as a significant reproducible increase in serum creatinine, urine protein or hematuria. Alternatively, or in addition, patients can be monitored for levels of antinuclear antibodies and antibodies to double stranded DNA. Treatment for SLE includes high dose corticosteroids and / or cyclophosphamide (HDCC). For systemic lupus erythematosus, patients can be monitored for levels of antinuclear antibodies and antibodies to double-stranded DNA.

  A particular aspect of the present invention is the treatment of lupus nephritis using a combination of an immunosuppressive drug and a BLyS antagonist such as atacicept.

  Spondyloarthropathies are a group of joint disorders such as ankylosing spondylitis, psoriatic arthritis and Crohn's disease. The success of treatment can be determined by an effective patient and physician-wide assessment measurement tool.

  A further autoimmune disease that can be treated using the methods of the present invention is psoriasis. A variety of medications are currently used to treat psoriasis; treatment varies in direct relation to disease severity. Patients with milder forms of psoriasis typically use local treatments such as topical steroids, anthralin, calcipotriene, clobetasol and tazarotene to manage the disease, while moderate and severe Patients with psoriasis are likely to use systemic (methotrexate, retinoid, cyclosporine, PLTVA and UVB) therapy. Tar is also used. These therapies have a combination of safety issues, time-consuming regimens or tedious treatment processes. In addition, some require expensive equipment and dedicated space in an office setting. Systemic medications can cause serious side effects such as antihypertension, hyperlipidemia, myelosuppression, liver disease, kidney disease and gastrointestinal upset. Furthermore, the use of phototherapy can increase the incidence of skin cancer. In addition to the inconvenience and discomfort associated with the use of local therapy, phototherapy and systemic therapy require patients to be periodically turned on and off to monitor lifetime exposure for their side effects .

Therapeutic efficacy for psoriasis includes clinical signs of the disease, including changes in the physician's overall rating (PGA) as compared to baseline symptoms, and the psoriasis area severity index (PASI) score, psoriasis symptom rating (PSA) Assessed by monitoring changes in symptoms. Patients can be measured periodically throughout treatment with respect to a visual analog scale used to indicate the degree of pruritus experienced at a particular time.
Dose administration

  Due to the indications to be treated and factors related to dose administration well known to those skilled in the art, the BLyS antagonists and immunosuppressive agents of the present invention treat their indications with minimal toxicity and side effects. Is administered at a dosage that is effective. In general, the BLyS antagonists of the present invention are administered to human patients in a dosage range of about 0.25 mg / kg body weight to about 25 mg / kg, preferably about 1 mg / kg to about 10 mg / kg. In other words, the preferred dosage range for BLyS antagonists is about 75 to about 190 mg / dose administration. In one preferred embodiment, the dosage is about 150 mg / dose for a BLyS antagonist.

  The therapeutic methods of the present invention comprise a combination of simultaneous and sequential administration of an anti-CD20 agent and a BLyS antagonist (both referred to herein as a therapeutic moiety). For sequential administration, the therapeutic moieties can be administered in any order, i.e. first anti-CD20 followed by a BLyS antagonist. A patient can be treated with an agent after being treated with the agent and monitored for efficacy. For example, if an anti-CD20 agent produces a partial response, treatment is passed on to a BLyS antagonist to achieve a full response (and vice versa). Alternatively, the patient may be administered both drugs first and then only one or the other drug.

  Mammals in need thereof to tolerate the drug and / or condition the patient to reduce the occurrence of side effects such as infusion related symptoms resulting from the initial and subsequent administration of the therapeutic compound Is administered one or both drugs in the initial or initial conditioning dose, and then one or both drugs in the second therapeutically effective dose of the drug, in which case the second and subsequent The dose is higher than the initial dose. The initial dose serves to condition the mammal and tolerate a higher second therapeutic dose. In this way, mammals can tolerate higher doses of therapeutic compounds that can be administered initially. A “conditional dose” is a dose that reduces or reduces the frequency or severity of initial dose side effects associated with administration of a therapeutic compound. The conditioning dose can be a therapeutic dose, a subtherapeutic dose, a symptomatic dose or a subsymptomatic dose. A therapeutic dose is a dose that exhibits a therapeutic effect on a patient, and a sub-therapeutic dose is a dose that does not exhibit a therapeutic effect on a treated patient. A symptom dose is a dose that induces at least one side effect on administration, and a subsymptom dose is a dose that does not induce side effects. Some side effects are fever, headache, nausea, vomiting, dyspnea, muscle pain and chills.

In addition to the conditioning dose regimen, there are a number of other regimens that can be followed to achieve the disease relief of the present invention. One such approach is treatment with a short course of immunosuppressive drugs followed by tapering followed by a combination of anti-CD20 and BLyS antagonists. For example, corticosteroids can be administered 1000-1500 mg orally twice daily for 4 weeks, then gradually decreasing from 5 mg / week to 10 mg / day over the course of 10 weeks. Once a BLyS antagonist and / or anti-CD agent has been initiated, a loading dose regimen may be appropriate. In particular, atacicept can be administered twice a week for 4 weeks, followed by weekly administrations for long periods, for example 48 weeks. If the patient's white blood cell count remains at an acceptable level (ie, approximately 3.0 x 10 ⁹ / L or 3000 / mm ² ), the dose should be increased weekly to a maximum dose of 1000 mg 3 times / day. obtain.
Route of administration

BLyS antagonists and anti-CD agents can be administered subcutaneously, intramuscularly, intraperitoneally, intracerebral and spinal cord, intraarticular, synovial fluid according to known methods, for example by intravenous administration, for example as a bolus, or by continuous infusion over time. It is administered to human patients by intracapsular, intrathecal or inhalation routes. BLyS antagonists with appropriate formulations can also be administered orally or topically. BLyS antagonists and anti-CD20 agents are generally administered by intravenous or subcutaneous administration. Different therapeutic moieties can be administered by the same or different routes.
Products and kits

  Another embodiment of the invention is a product comprising a BLyS antagonist and an anti-CD20 agent for treating a B cell regulatory autoimmune disorder as described above. In one specific example, the product contains Actacicept and Rituxan® for the treatment of SLE.

  The product includes at least one container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition of the invention that is effective for treating the condition and has a sterile access port (eg, the container is an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle. obtain). The label or package insert indicates that the composition is used to treat a specific condition such as lupus nephritis or rheumatoid arthritis. The label or package insert further includes a device for administering the composition to the patient. Additionally, the product may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles and syringes.

  Kits are also provided that are useful for various purposes, eg, for B cell killing assays. When using the product, the kit includes a container and a label or packaging insert on or associated with the container. The container holds a composition comprising at least one immunosuppressive drug of the invention and one BLyS antagonist. For example, additional containers containing diluents and buffers, control antibodies can be included. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

Example 1
Production of BLyS antagonists Four amino-terminal truncated versions of TACI-Fc are generated. All four have a modified human tissue plasminogen activator signal sequence (SEQ ID NO: 41) as disclosed in WO 02/094852 fused to amino acid residue 30 of SEQ ID NO: 2. However, the four proteins differed in the position of Fc5 fused to the TACI amino acid sequence of SEQ ID NO: 2. Table 1 abbreviates the structures of the four fusion proteins.

Protein-encoded expression cassettes were generated by overlapping PCR using standard techniques (see, eg, Horton et al., Gene 77: 61 (1989)). A nucleic acid molecule encoding TACI and a nucleic acid molecule encoding Fc5 were used as PCR templates. Oligonucleotide primers are identified in Tables 2 and 3.

  The initial PCR amplification consisted of two reactions for each of the four amino-terminal truncated versions. For each version, the two reactions were performed separately, with 5 'and 3' TACI oligonucleotides in one reaction and 5 'and 3' Fc5 oligonucleotides in another reaction. The conditions for the initial PCR amplification are shown below. In a final volume of 25 μl, about 200 ng template DNA, 2.5 μl 10 × Pfu reaction buffer (Stratagene), 2 μl 2.5 mM dNTP, 0.5 μl 20 μM each 5 ′ and 3 ′ oligonucleotide, and 0.5 μl of Pfu polymerase (2.5 units, Stratagene) was added. The amplification temperature profile consisted of 35 cycles of 94 ° C. for 3 minutes; 94 ° C. for 15 seconds, 50 ° C. for 15 seconds, 72 ° C. for 2 minutes; then 72 ° C. for 2 minutes. The reaction products were fractionated by agarose gel electrophoresis, the band corresponding to the predicted size was excised from the gel and recovered using the Qiagen QIAQUICK gel extraction kit (Qiagen) according to the manufacturer's instructions.

  A second round of PCR amplification or duplicate PCR amplification reaction was performed using the gel purified fragment from the first PCR as the DNA template. The conditions for the second PCR amplification are shown below. In a final volume of 25 μl, approximately 10 ng of template DNA (each TACI fragment and Fc5 fragment), 2.5 μl of 10 × Pfu reaction buffer (Stratagene), 2 μl of 2.5 mM dNTP, 0.5 μl of 20 μM each ZC24, 903 (SEQ ID NO: 27) and ZC24,946 (SEQ ID NO: 30), and 0.5 μl of Pfu polymerase (2.5 units, Stratagene) were added. The amplification temperature profile consisted of 35 cycles of 94 ° C for 1 minute; 94 ° C for 15 seconds, 55 ° C for 15 seconds, 72 ° C for 2 minutes; then 72 ° C for 2 minutes. The reaction products were fractionated by agarose gel electrophoresis, the band corresponding to the predicted size was excised from the gel and recovered using the Qiagen QIAQUICK gel extraction kit (Qiagen) according to the manufacturer's instructions.

Each of the four versions of the amino-terminal truncated TACI-Fc PCR product was cloned separately using Invitrogen's ZEROBLUNT TOPO PCR cloning kit according to the manufacturer's recommended protocol. Table 4 identifies the nucleotide and amino acid sequences of these TACI-Fc constructs.

  After verification of the nucleotide sequence, each of the four versions of the amino-terminal truncated TACI-Fc fusion was digested with FseI and AscI to release the amino acid coding segment. The FseI-AscI fragment was ligated into a mammalian expression vector containing a CMV promoter and an SV40 polyA segment. The expression vector was introduced into Chinese hamster ovary cells as described below.

Example 2
Production of TACI-Fc protein by Chinese hamster ovary cells TACI-Fc expression constructs were used to transfect suspension-adapted Chinese hamster ovary (CHO) DG44 cells grown in animal protein-free medium by electroporation. (Urlaub et al., Som. Cell. Molec. Genet. 12: 555 (1986)). CHO DG44 cells lack a functional dihydrofolate reductase gene due to deletions at both dihydrofolate reductase chromosomal locations. Growth of cells in the presence of increased concentrations of methotrexate results in amplification of the dihydrofolate reductase gene as well as the linked recombinant protein-encoding gene on the expression construct.

CHO DG44 cells are subcultured in a PFCHO field position (JRH Biosciences, Lenexa, KS), 4 mM L-glutamine (JRH Biosciences) and 1 × hypoxanthine-thymidine supplement (Life Technologies), and a rotating shaker Cells were incubated at 37 ° C., 5% CO ₂ in the above 120 RPM Corning shake flask. Cells were separately transfected with a linearized expression plasmid. To ensure sterility, 200 μg of plasmid DNA in an Eppendorf tube was added to 20 μl of sheared salmon sperm carrier DNA (5 ′ → 3 ′ Inc. Boulder, CO, 10 mg / ml), 22 μl of 3 M NaOAc (pH 5 .2) and a koto combined with 484 μl of 100% ethanol (Gold Shield Chemical Co., Hayward, Calif.), A single ethanol precipitation step was performed on ice for 25 minutes. After incubation, the tubes were centrifuged at 14,000 RPM in a microcentrifuge placed in a 4 ° C cooling chamber, the supernatant was removed, the pellet was washed twice with 0.5 ml 70% ethanol and allowed to air dry .

CHO DG44 cells were prepared while drying the DNA pellet by centrifuging 10 ⁶ total cells (16.5 ml) in a 25 ml conical centrifuge tube at 900 RPM for 5 minutes. CHO DG44 cells were resuspended in PFCHO growth medium with a total volume of 300 μl and placed in a Gene-Pulser cuvette (Bio-Rad) with a 0.4 cm electrode gap. After a drying time of about 50 minutes, the DNA is resuspended in 500 μl PFCHO growth medium, added to the cells in the cuvette so that the total volume does not exceed 800 μl, and left at room temperature for 5 minutes, Reduced formation. The cuvette was placed in a Bio-Rad Gene Pulser II unit set at 0.296 kV (kilovolt) and 0.950 HC (high electric capacity) and immediately electroporated.

Cells were incubated at room temperature for 5 minutes before being placed in a total volume of 20 ml PFCHO medium in a CoStar T-75 flask. Place the flask for 48 hours at 37 ° C. with 5% CO ₂ and then count the cells with a hemacytometer utilizing trypan blue exclusion and contain no hypoxanthine-thymidine supplement, 200 mM methotrexate (Cal Biochem) In PFCHO selection medium containing

  At the time of recovery of the methotrexate selection process, conditioned media containing secreted TACI-Fc protein was examined by Western blot analysis.

方法
試料 Example 3
Immunophenotyping of B cell subsets in cynomolgus monkey peripheral blood and spleen cells The following combinations of markers were used to detect cell populations:

Method Sample

Species / lineage and source were cynomolgus monkeys supplied by Novaprim Group, Mauritius. The matrix used was peripheral whole blood (in 8% EDTA solution as an anticoagulant with freshly collected spleen tissue).
Monoclonal antibody

Commercial human monoclonal antibodies that cross-react with cynomolgus monkey antigens as reported in related papers were used (Table 1 below).
Test procedure Blood

  Cell staining was performed by adding 100 μL of whole blood to an appropriate amount of antibody cocktail (Table 1). The antibody / blood mixture was mixed well and then incubated at 4 ° C. for 15-20 minutes. At the end of the incubation period, add 1 mL of BD FACLYse lysis solution (at 1:10 dilution), mix well by vortexing for a few seconds at low / moderate speed, then in the dark at room temperature Incubated for about 12 minutes. At the end, 2 mL of staining buffer (PBS; 2% FBS and 10 mM NaN3) was added to the mixture. The tube was capped again with the original cap, mixed upside down, then placed in a rocking bucket and centrifuged at 250 xg for 6-7 minutes at room temperature. The supernatant was decanted and a second wash was performed. At the end of the second wash step, 100 μL of cytofix buffer (BD) was added to the cell pellet and mixed well by vortexing at low / moderate speed. Samples were kept at 4 ° C. in the dark until data acquisition. Samples were resuspended in staining buffer immediately prior to acquisition on a FACSCalibur cytometer.

Blood samples (100 μL) were washed twice with 1 mL staining buffer for surface immunoglobulin staining. The supernatant was aspirated and 10 μg / μL of rabbit or goat immunoglobulin (for IgD and IgM staining, respectively) was added and incubated at 37 ° C. for 30 minutes. At the end of the blocking phase, sample staining and lysis were performed as described above.
Splenocytes

  Spleen tissue was placed in a Petri dish containing chilled medium (DMEM, 2% FCS, 2 mM EDTA) and carefully minced with a scalpel to obtain a cell suspension. Cells were broken apart by pipetting up and down with a syringe, filtered through a 70 μm nylon mesh, washed and adjusted to 107 / mL in DMEM. Mouse IgG (5-10 μg / 106 cells) was added to the cell suspension and incubated on ice for 5-10 minutes to block nonspecific binding sites.

The spleen cells were then stained by adding 100 μL of cynomolgus spleen cells to the appropriate amount of antibody. The antibody / cell mixture was mixed and then incubated at 4 ° C. for 15-20 minutes. At the end of the incubation period, 1 mL of ammonium chloride lysis solution was added. Samples were mixed thoroughly by swirling for several seconds at low / moderate speed and incubated at room temperature for about 7 minutes in the dark. Next, 2 mL of staining buffer (PBS; 2% FBS, 10 mM NaN3) was added. The tube was capped again with the original cap, mixed upside down, then placed in a rocking bucket and centrifuged at 250 xg for 6-7 minutes at room temperature. The supernatant was decanted and the washing procedure was repeated. At the end of the second wash step, 100 μL of cytofix buffer (BD) was added to the cell pellet and mixed well by vortexing on a low / moderate speed vortex mixer. Samples were kept at 4 ° C. in the dark until data acquisition. Samples were resuspended in staining buffer immediately prior to acquisition on a FACSCalibur cytometer.
Flow cytometry analysis

  FACSCalibur flow cytometry (BD Biosciences) equipped with 488 nm and 633 nm laser lines and CellQuest software (BD Biosciences) were used for acquisition and analysis of all samples. Initially, CaliBriteBeads (BD Biosciences) was run to check for proper instrument function and detector linearity. The correction control was moved to validate the instrument settings.

The lymphocyte acquisition gate was set with forward / side scatter parameters to prove that it contains lymphocytes predominantly. For each sample, a minimum of 20000 events in the lymphocyte gate were acquired.
Results Blood stain

Human antibodies that cross-react with cynomolgus monkey cells as reported in the literature (Vugmeyster Y. et al., 2004, Yoshino N. et al., 2000) were used at concentrations recommended by the manufacturer (Table 5). .

  Several antibodies were pretested to determine the best applicable dilution. In addition, the isotype control combination was moved to examine the presence of non-specific fluorescence and to set a quadrant for each sample. Acceptable results were obtained for all markers.

In order to identify naïve (CD40 + CD21 + CD27−) and memory (CD40 + CD21 + CD27 +) B cells, it was essential to use conjugated CD27−PE, otherwise a double positive population CD21 + CD27 + could not be observed. FITC modification of antibodies can sometimes suppress antibodies that bind monkey cells (Reimann et al., 1994). The gating strategy that is subsequently performed to analyze the data is shown below (FIG. 1). To examine the quality of staining, blood samples were pre-experimented with the ADVIA 120 Hematology System (Bayer Diagnostics) and the resulting lymphocyte percentage (%) was used for lymphocyte gate percentage comparison.
Splenocytes

細胞集団の分析のためにその後に実行されるゲーティング戦略を、図１Ａ、１Ｂおよび１Ｃに例示する。
参考文献 Monoclonal antibodies used for blood staining were also employed for splenocyte staining. Different dilutions were first tested to optimize the concentration. A summary of antibodies and selected concentrations is given below:

Subsequent gating strategies performed for the analysis of the cell population are illustrated in FIGS. 1A, 1B and 1C.
References

  Vugmeyster Y., Howell K., Bakshi A., Flores C, Hwang O., McKeever K., (2004); B-cells subsets in blood and lymphoid organs in Macaca facsicularis, Cytometry, Part A 6 IA: 69-75 .

  Yoshino N., Ami Y., Terao K., Tashiro F., Honda M., (2000); Upgrading of flow cytometric analysis for absolute counts, cytokines and other antigenic molecules of Cynomolgus Monkeys (Macaca fascicularis) by using anti-human cross-reactive antibodies, Exp. Anim. 49 (2): 97- 110.

  Reimann KA, Waite B., Lee-Parritz DE, Lin W., Uchanska-Ziegler B., O'Connell MJ., Letvin NL, (1994); Use of human leucocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of Rhesus Monkeys , Cytometry, 17: 102-108.

Example 4
Combination of atacicept and rituxan in cynomolgus monkeys Atacicept and anti-CD20 monoclonal antibody (Rituxan®) co-administration were tested in a 31-week study in cynomolgus monkeys.

  Rituximab was administered as a slow intravenous infusion (200 mg / hr) via the tail vein at a dose of 20 mg / kg in a volume of 2 mL / kg, once per week for 4 weeks. A control group for rituximab received vehicle (0.9% sodium chloride solution) instead of rituximab.

  Atacicept was administered as a subcutaneous injection twice weekly into the quadriceps muscle (using alternating sites) at a dose of 20 mg / kg in a volume of 1 mL / kg for 13 weeks. A control group for atacicept was administered with a vehicle (PBS1 × pH 7.2) in place of atacicept.

  Animals were sacrificed at different time points during the study for evaluation of pharmacological effects, gross lesions and histopathology. The treatment period for selected animals was followed by a 14 week recovery period. The experimental design including the different combinations of the two compounds, the number of animals per group and the planned slaughtered animals is shown in the following table (Table 7).

  FACS analysis was performed on splenocytes as described in Example 2 above. Analysis was performed on the spleen at 5 weeks (for some groups only) and 18 weeks (for all groups), thus limiting the analysis to only 18 weeks. The data for the 18th week are attached as Table 8 below.

結論 Of the total splenocytes at 18 weeks, approximately 41% are CD40 + B cells in Group 1 (vehicle) and 2 (rituximab alone) animals. In contrast, after treatment with atacicept at 18 weeks (group 3), only 15% of the splenocytes are CD40 + B cells. When animals are treated sequentially with rituximab and then with atacicept (group 4), at week 18 only 6% of splenocytes are CD40 + B cells. This 35% reduction in Group 4 animals is a greater additive effect than atacicept alone (−27%) and rituximab alone (−1%). A similar larger additive decrease is observed for memory B cells (% of CD40 + splenocytes being CD21 + / CD27 +).
Table 8: Flow cytometric immunophenotyping of total B lymphocytes (as CD40 + lymphocyte%) and memory B lymphocytes (% of CD40 + spleen cells that are CD21 + / CD27 +) in the spleen

Conclusion

  The experiments herein show that the combination of anti-CD20 agents and BLyS antagonists such as Rituxan® and atacicept is synergistic at the B cell level compared to the level of reduction by Rituxan® and BLyS antagonist alone. An unexpected result was proved in that it caused a depletion of the environment.

Example 5 Effect of combined treatment with atacicept and rituximab in human CD20 transgenic mice Example 5
Rituximab does not bind to mouse CD20 and therefore cannot be used to deplete B cells in normal mice. Furthermore, there are no commercially available anti-mouse CD20 mAbs effective for in vivo B cell depletion. Gong et al. (J. Immunol 174: 817-826; 2005) reported that B was treated with anti-human CD20 mAbs (rituximab and ocrelizumab) as well as those observed in humans treated with these mAbs. A strain of human CD20 transgenic mice (generated using bacterial artificial chromosomes / BAC technology) that induce cell depletion was utilized. They also showed that combining anti-CD20 mAb therapy with BR3-Fc (BAFFR-Ig, a BLyS single inhibitor) significantly enhanced splenic B cell depletion. This example utilizes a strain of hCD20 Tg mice obtained with permission from Yale University Mark Shlomchik (Ahuja et al., J. Immunol 179: 3351-3361; 2007).

用量調製 General approach: Use the optimal dose of each therapeutic agent, then combine the two in each order (first rituximab or first atacicept) and assess B cell depletion. B cell depletion is assessed at the optimal time point (3 weeks), but includes an additional group to assess B cell recovery after treatment cessation (~ 20 weeks).
Materials and methods

Dose preparation

Following the schedule and route for the atacicept treatment group, phosphate buffered saline (PBS; Gibco) was administered subcutaneously at 0.2 ml / animal three times a week. Atacicept was thawed at room temperature, mixed gently but well, then diluted in room temperature PBS for subcutaneous injection of 0.2 mL or less / mouse. Rituximab was used undiluted and supplied at 10 mg / mL.
Animal management, acclimatization and containment

Room temperature was maintained at 70-74 ° F. and humidity was maintained at 30% -70%. A 12 hour light / dark cycle was used, however, room light may be lit during the dark cycle to accommodate test related activities. Each animal had free access to rodent food (irradiation 5056, Pico Lab, Richmoud, IN) and water. The procedure used for this study is intended to avoid or minimize discomfort, distress or pain to the animals. The treatment of test animals follows the conditions specified in the guidelines for the management and use of laboratory animals (ILAR publication, 1996, National Academy Press). Animals were randomly assigned to various treatment groups as detailed in Table 10.

Treatment group: The dose administration period is called the dose administration phase. The first day of administration is called “Day 1”. The day before the first day is “−1 day”. The day after the final sacrifice in the dose administration phase is the first day of the recovery phase.

Rituximab was administered intraperitoneally (IP). Atacicept and vehicle (PBS) were administered three times a week (9 doses total) by subcutaneous (SC) injection in the subscapular region. The initial dose is administered on day 1 (D1). In the combination treatment group, and where relevant, all animals received an IP injection first, followed by a SC injection within a 60 minute period. Group 4: first rituximab, then atacicept; group 5: first atacicept, then rituximab. The dose volume was adjusted weekly according to individual animal body weight.
Summary of study endpoints:
Whole blood (150 μL; EDTA) was collected and flow cytometric analysis was performed on T and B cell subsets (see Table 16).
Serum (˜100 μL whole blood in serum separation tubes) is collected at various time points and then analyzed for total IgG ₁ , IgG _2a , IgM, IgE and IgA by Luminex assay.
・ At present, about half of the animals are slaughtered. The rest is sacrificed at about 20 weeks of testing.
At slaughter, spleens were collected and processed for flow analysis and subsequent IHC / histology. Before blood collection, splenectomy was performed under isoflurane anesthesia. Whole spleen was weighed and recorded before being sectioned.
At the time of sacrifice, major peripheral lymph nodes (groin, axilla, upper arm, neck and mesentery) were collected for possible future IHC and histology. Lymph nodes were fixed with formalin or zinc tris buffer and stored in 70% alcohol for possible future use.
• For histological examination: 1/3 of the spleen and one LLN (left LN) were placed in zinc tris and the same tissue (right LN) was taken in 10% NBF.

All animals in Groups 1-5 (n = 53) received serum (100 μL blood in serum separator tubes) and whole blood (150 μL in EDTA tubes) via the retro-orbital vein on day -5. ).
Serum collection: A minimum of 100 μL of whole blood was placed in a serum separator tube and allowed to clot for a minimum of 15 minutes. The blood was then spun at 4000 rpm for 10 minutes. A minimum of 50 μL of serum was dispensed into the second container. The resulting aliquot was stored at ≦ 60 ° C.

  Whole blood in EDTA collection: A minimum of 150 μL of blood was placed in a micro blood collection tube containing EDTA. The tube was gently inverted a minimum of 20 times. Whole blood in EDTA samples was stored at room temperature until processed for flow cytometry.

All sacrificed animals were anesthetized with isoflurane. All sacrificed animals were collected from blood serum, spleen and major peripheral lymph nodes. After the spleen was collected at the isoflurane anesthesia department, a blood sample was taken to avoid changes in the spleen cell subset. The whole spleen was weighed. The spleen was cut into three sections (head, middle, tail) and processed into the table shown in Table 12.

Lymph node collection: Major peripheral lymph nodes (groin, axilla, upper arm, cervix and mesentery) were collected for histology / IHC in cassettes (see Table 13).
a. The cassette containing the left lymph node (except for the mesenteric LN) for IHC is placed in zinc tris buffer.
b. A cassette containing the right lymph nodes (except for the mesenteric LN) for histology is placed in 10% NBF.
c. The cassette containing the mesenteric lymph nodes is collected in 10% NBF by collecting the entire intestine (from the stomach to just above the rectum) without washing.
All samples were stored at room temperature.

¹ 表現型［Ｂ２２０＋／ＣＤ１９＋／ｈｕＣＤ２０−］は、Ｂ２２０およびネズミＣＤ１９表面抗原をともに発現するが、ヒトＣＤ２０導入遺伝子を発現しないＢ細胞の集団を説明する。
² 表現型［Ｂ２２０＋／ＣＤ１９＋／ｈｕＣＤ２０＋］は、Ｂ２２０およびネズミＣＤ１９表面抗原を、ヒトＣＤ２０導入遺伝子を含めて発現するＢ細胞の集団を説明する。 Whole blood immunophenotyping: In summary, whole blood was collected in a BD Microtainer ^™ tube containing K ₂ EDTA anticoagulant. A 50 μL aliquot of whole blood was incubated with an appropriate working antibody cocktail (see Table 14) to lyse erythrocytes. Prior to sample acquisition in flow cytometry, Flow Count ^™ fluorescent microspheres are added to each sample tube to calculate absolute cell concentration. Data acquisition was performed on a BD FACSCalibur flow cytometer equipped with a 15 mW air-cooled argon ion laser (488 nm emission) and a red diode laser (635 nm emission). Instrument calibration is performed every day of sample acquisition and appropriate controls are used to verify fluorescence correction and population gating. In general, there are two or more blood collection time points in the test of the present invention.

^{The 1} phenotype [B220 + / CD19 + / huCD20−] describes a population of B cells that express both B220 and murine CD19 surface antigen but do not express the human CD20 transgene.
^{The 2} phenotype [B220 + / CD19 + / huCD20 +] describes a population of B cells that express B220 and murine CD19 surface antigens, including the human CD20 transgene.

ＩＨＣ分析 Spleen immunophenotyping: Briefly, single cells were isolated and incubated with the appropriate antibody cocktail (see Table 15). Instrument calibration and data acquisition were performed as in the case of whole blood immunophenotyping.

IHC analysis

  Tissue: Test tissue includes samples from both spleen and lymph nodes. For spleen samples, transverse sections of the spleen (head and tail pieces) from each animal are included. Splenic head sections (zinc tris-fixed) are stained with rat monoclonal antibodies against CD45R / B220, CD138 or CD5 alone. A subset of tissue sections are stained with rat isotype IgG as a negative control. Spleen tail sections (formalin fixed) are stained with biotinylated PNA (if GC needs to be visualized; H & E is sufficient) and H & E. A subset of tissue sections is also stained with biotinylated parathyroid hormone related protein (PTHrP) as a negative control.

  Test lymph nodes from each animal include the groin, axilla, upper arm, neck and mesenteric lymph nodes. Lymph nodes are fixed with formalin or zinc tris and kept in 70% alcohol for possible future use.

  Antibody: As an antibody to be used, mouse CD45R / B220 (clone RA3-6B2, isotype: rat IgG2a, k; 0.5 mg / mL, # 557390, BD Biosciences, San Jose, Calif.), CD5 (Ly-1, clone 53) -7.3, 0.5 mg / mL, # 553017, BD Biosciences), and three rat monoclonal antibodies against CD138 (clone Syndecan-1, 0.5 mg / mL, # 553712, BD Biosciences).

Statistical analysis: Statistical analysis of group differences in organ weight, B cell count and immunoglobulin levels was performed using analysis of variance (ANOVA).
result

^* 太字数字／影付セルは、この薬剤組合せに関して付加的作用より大きいものを示す。
Table 16 represents the% change in absolute concentration of B cells in mouse peripheral blood samples at various time points for the standard treatment group.

^* Bold numbers / shaded cells indicate greater than additional effects for this drug combination.

^* 太字数字／影付セルは、この薬剤組合せに関して付加的作用より大きいものを示す。
結論 Table 17 specifically shows an alternative method of expressing the same data as changes in the presence of positive lymphocytes or B220 plus cells. This is encompassed in a manner similar to that reported the results of the cynomolgus monkey experiment of Example 4 (required for the use of cells isolated from the spleen rather than peripheral blood).

^* Bold numbers / shaded cells indicate greater than additional effects for this drug combination.
Conclusion

The experiments herein show that the combination of an anti-CD20 agent and a BLyS antagonist, such as Rituxan® and atacicept, at multiple time points compared to the level of reduction by Rituxan® and BLyS antagonist alone, An unexpected result has been demonstrated in that it results in synergistic depletion at the B cell level.
References

  References cited within this application are hereby incorporated by reference, including patents, published applications and other publications.

Claims (19)

  A method for reducing the number of B cells in a mammal comprising administering a BLyS antagonist comprising a polypeptide comprising the sequence of SEQ ID NO: 26 and an anti-CD20 agent.   2. The method of claim 1, wherein the BLyS antagonist and the anti-CD20 agent act synergistically to reduce B cell levels.   The method of claim 2, wherein the reduced B cell level is the number of memory B cells.   The method of claim 1, wherein the anti-CD20 agent is selected from the group consisting of Rituxan®, ocrelizumab, ofatumumab, TRU-015 and DXL625.   A method of alleviating a B cell regulatory autoimmune disorder, wherein a therapeutically effective amount of a BLyS antagonist comprising a polypeptide comprising the sequence of SEQ ID NO: 26 and an anti-CD20 agent is administered to a patient suffering from said disorder A method involving that.   6. The method of claim 5, wherein the anti-CD20 agent is selected from the group consisting of Rituxan®, ocrelizumab, ofatumumab, TRU-015 and DXL625.   The method of claim 6, wherein the anti-CD20 agent is Rituxan®.   6. The method of claim 5, wherein the BLyS antagonist and the anti-CD20 agent act synergistically to reduce B cell levels.   9. The method of claim 8, wherein the reduced B cell level is the number of memory B cells.   The autoimmune diseases are rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic Thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM multiple neuritis, myasthenia gravis, vasculitis, diabetes mellitus, Raynaud's syndrome, Sjogren's syndrome and 6. The method of claim 5, wherein the method is selected from the group consisting of glomerulonephritis.   11. The method of claim 10, wherein the autoimmune disease is systemic lupus erythematosus (SLE).   The method of claim 11, wherein the anti-CD20 agent is Rituxan®.   7. The method of claim 6, wherein the BLyS antagonist is administered at a dosage of about 1 to about 25 mg / kg and the anti-CD20 is administered at a dosage of about 1 to about 25 mg / kg.   14. The method of claim 13, wherein the BLyS antagonist is administered at a dose of about 20 mg / kg and anti-CD20 is administered at a dose of about 20 mg / kg.   Cyclophosphamide (CYC), azathioprine (AZA), cyclosporin A (CSA), mycophenolate mofetil (MMF), nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, prednisone and disease modifying anti-rheumatic drugs ( 7. The method of claim 6, wherein the BLyS antagonist and the anti-CD20 agent are administered in combination with a therapy using an immunosuppressive drug selected from the group consisting of DMARD).   A composition comprising a BLyS antagonist comprising a polypeptide comprising the sequence of SEQ ID NO: 26 and an anti-CD20 agent.   The composition according to claim 16, wherein the anti-CD20 agent is Rituxan®.   A product comprising a BLyS antagonist and anti-CD20 agent comprising a polypeptide comprising the sequence of SEQ ID NO: 26, and a label, wherein the label is for the treatment of a B cell regulatory autoimmune disorder Indicating the product.   The product of claim 18, wherein the anti-CD20 agent is Rituxan®.

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